Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Activation of TLR2 is dependent on PPAD expression and activity. U251 MG cells overexpressing the TLR2 receptor were infected (MOI=100) or treated for 4 h with (A) ATCC 33277 strain or Pam3CSK4 (1 μg/ml), n=5-7; (B) various P. gingivalis strains and ATCC-derived isogenic mutants of catalytically inactive PPAD (C351A PPAD), n=3-5; (C) ATCC 33277 wild-type and PPAD mutant strains and sonicates (cells were lysed by sonication (sonic.)), n=4; or (D) outer membrane vesicles (OMVs) isolated from ATCC 33277 strain and its isogenic PPAD mutants (2 µg/ml), n=4. Results in (A) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Results in (B–D) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Data are normalized to those from cells stimulated/infected with the same factor and transfected with an empty vector. Mean + SD; ****p < 0.0001; ***p < 0.001; ns, no statistical significance; 1-way ANOVA both followed by Tukey’s multiple comparisons test. ATCC WT- ATCC 33277.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Expressing, Activity Assay, Infection, Derivative Assay, Mutagenesis, Sonication, Membrane, Isolation, Luciferase, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Fimbriae purified from the wild-type ATCC 33277 strain activate the TLR2 receptor. U251 MG cells overexpressing TLR2 were: (A) infected for 4 h with WT ATCC 33277 and its isogenic major fimbriae (delFimA) mutant strain (MOI=100), n=4-5 (B) infected for 4 h with various ATCC 33277-derived PPAD and FimA mutants as well as the WT W83 strain at different MOI (all strains MOI=20-100 with an additional MOI=5 for WT ATCC 33277), n=3 or (C) infected for 2, 4 or 6 h with WT ATCC 33277 or the PPAD mutant strains (MOI=100), n=3; and (D) treated for 4 h with purified fimbriae (10 µg/ml) from WT ATCC 332771 (FimA WT) or the PPAD mutant (FimA delPPAD) strains; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (E) HEK Blue cells overexpressing TLR2 were treated with purified fimbriae (10 µg/ml) isolated from the WT ATCC 33277 strain (FimA WT) or the PPAD mutant (FimA delPPAD), n=3. Results are presented as the mean ± SD fold induction compared to control (unstimulated) cells. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. In (B) each condition was compared to control uninfected cells and in (C) comparisons were performed for each timepoint separately and significant differences compared to WT ATCC 33277 are depicted in the graph.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Purification, Infection, Mutagenesis, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Isolation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Both active PPAD and fimbriae are crucial for activation of TLR2. U251 MG cells overexpressing the TLR2 receptor were infected for 4 h (MOI=100) with various ATCC 33277-derived isogenic mutants expressing different forms of PPAD (the T1 form, the T2-hyperactive form, and the catalytically inactive C351A mutant form) or FimA; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. ****p < 0.0001; ns, no statistical significance; 1-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Infection, Derivative Assay, Expressing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: The ability of clinical strains to activate TLR2 is much weaker than that of ATCC 33277. (A) Cells were infected for 4 h (MOI=100) with various clinical strains (k1-k10), ATCC 33277 (ATCC WT), or W83, n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (B) Western blot analysis of laboratory and clinical strain cultures (adjusted to OD 600 ) to detect FimA. (C) PPAD activity in whole laboratory and clinical strain cultures (adjusted to OD 600 ), n=6. Results were compared with those from the ATCC 332777 strain. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) The FimA type determined by sequencing of the fimA gene in clinical strains.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Sequencing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 1. Generation of human Tspan15-expressing MEFs as an immunogen and validation of resulting mouse anti-human Tspan15 mAbs. (A) ADAM10-knockout MEFs (–) and ADAM10- knockout MEFs stably overexpressing FLAG-tagged Tspan15 (+) were lysed in 1% Triton X-100 lysis buffer and subjected to anti-FLAG (top panel) and anti-α-tubulin (bottom panel) western blotting. (B) Wild-type (WT) and Tspan15-knockout (KO) Jurkat human T cells were analysed by flow cytometry with tissue culture supernatant for each of the four mouse anti-human Tspan15 hybridomas (1C12, 4A4, 5D4 or 5F4; solid line), or with mouse IgG1 as a negative control (dotted line). Histograms are representative of two independent experiments. (C) HEK-293T cells were transfected with FLAG-tagged human TspanC8 expression constructs (except for Tspan10, which was of mouse origin) or an empty vector control (–),

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Biomarker Discovery, Knock-Out, Stable Transfection, Lysis, Western Blot, Flow Cytometry, Negative Control, Transfection, Construct, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 3. Tspan15 mAbs 1C12 and 4A4 partially inhibit ADAM10/Tspan15 activity. (Ai) Wild-type (WT), ADAM10-knockout (A10 KO) and Tspan15-knockout (T15 KO) HEK-293T cells were transfected with a VE-cadherin expression construct. Cells were treated with 10 μM DAPT to prevent post-ADAM10 proteolysis by γ-secretase, followed by 2 mM NEM for 30 minutes to activate ADAM10. Cells were lysed in 1% Triton X-100 lysis buffer and subjected to western blotting with an antibody against the cytoplasmic tail of VE-cadherin. No C-terminal fragment was detected in the absence of NEM (data not shown). (Aii) VE-cadherin cleavage data were quantitated to calculate the percentage cleaved. Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Dunnett’s multiple comparisons test (***p<0.001 compared to WT). Error bars represent standard error of the mean from three independent experiments. (B) Wild-type HEK-293T cells were transfected with VE-cadherin, treated with Tspan15 mAbs or MOPC-21 negative control mAb for 30 minutes, and stimulated with NEM as described for panel

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Activity Assay, Knock-Out, Transfection, Expressing, Construct, Lysis, Western Blot, Transformation Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 4. Tspan15 and ADAM10 co-localise on the cell surface. (Ai) A549 cells were fixed and stained with anti-ADAM10 mAb (red) and either anti-Tspan15 mAb 5D4 (green) or anti-CD9 mAb 1AA2 (green). ADAM10, Tspan15 and CD9 on the basal membrane were imaged using TIRF microscopy. Images shown are representative of 48 fields of view from four independent experiments (scale bar 10 µm). (Aii) The degree of co-localisation between ADAM10 and Tspan15 or CD9 was determined using Manders’ coefficients to measure the proportion of overlapping pixels contained within total ADAM10 signal in the red channel (M1) and total Tspan15 or CD9 signal in the green channel (M2). Data were arcsine- transformed and statistically analysed by a one-way ANOVA with a Tukey’s multiple comparisons test to compare M1 and M2, within and between Tspan15 and CD9 (***p<0.001 for all pairwise comparisons). Error bars represent standard error of the mean.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Staining, Membrane, Microscopy, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 5. ADAM10 is the principal Tspan15-interacting protein in HEK-293T cells. Wildtype (WT) and Tspan15-knockout (KO) HEK-293T cells were lysed in 1% digitonin lysis buffer and immunoprecipitated with Tspan15 mAb 1C12 cross-linked to protein G sepharose beads. Proteins were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Proteomic profiles of WT and Tspan15 KO HEK-293T immunoprecipitates are presented in a volcano plot to identify differentially expressed proteins. The minus log10 transformed p-value of each protein was plotted against the log2 transformed protein label free quantification ratio between the Tspan15 co-immunoprecipitation of WT samples and the control co-immunoprecipitation of Tspan15 KO samples. Proteins with significant fold change (p<0.05) are depicted in red; blue dots represent proteins with no significant changes in expression. A permutation-based false discovery rate estimation was applied and visualised as hyperbolic curves in grey.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Knock-Out, Lysis, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Quantitative Proteomics, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 6. Tspan15 protein expression requires ADAM10. (A) Tspan15 surface expression in wildtype (WT), Tspan15-knockout (KO) and ADAM10 KO Jurkat, HEK-293T and A549 cell lines were analysed

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 7. The requirement of Tspan15 for ADAM10 surface expression is cell type dependent. (A) ADAM10 surface expression in WT, ADAM10 KO and Tspan15 KO Jurkat, HEK-293T and A549 cells was measured by flow cytometry and quantitated as described in Figure 4A. (B) HUVECs were transfected with two different Tspan15 siRNAs or negative control siRNA and surface expression of ADAM10 was measured by flow cytometry and analysed as described in Figure 6A.

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Expressing, Flow Cytometry, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 8. ADAM10 and Tspan15 form dynamic bimolecular fluorescence complementation (BiFC) complexes. (A) Schematic representation of ADAM10 tagged with the C-terminal half of superfolder GFP (sfGFP-C), Tspan15 tagged with the N-terminal half of superfolder GFP (sfGFP-N) and the predicted ADAM10/Tspan15 BiFC dimer. Solid ovals represent N-glycosylation. (B) HEK-293T cells were transfected with the ADAM10 and Tspan15 BiFC expression constructs, fixed and stained with Alexa Fluor® 647-conjugated Tspan15 mAb 5D4, and analysed by confocal microscopy. The image shown is representative of middle plane sections taken from two independent experiments (scale bar 10 µm). (C-D) Fluorescence correlation spectroscopy (FCS) measurements from the upper membrane of HEK-293T expressing the ADAM10/Tspan15 BiFC complexes were used to determine the average particle concentration (C) and diffusion co-efficient (D) of the complexes. (E) Fluorescence fluctuations from the FCS reads were also subjected to photon counting histogram (PCH) analysis to obtain the average molecular brightness (ε) of particles within the confocal volume. The FCS data were separated into groups that preferentially fit to a one-component or a two-component PCH model with dimmer and brighter subcomponents. Data were obtained from 43 individual measurements from three independent experiments. Error bars represent standard errors of the mean, N is the number of particles, and cpm is the counts per molecule. Data were log-transformed and statistically analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test (***p<0.001).

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Fluorescence, Glycoproteomics, Transfection, Expressing, Construct, Staining, Confocal Microscopy, Spectroscopy, Membrane, Concentration Assay, Diffusion-based Assay, Transformation Assay

Journal: Journal of Biological Chemistry

Article Title: The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

doi: 10.1074/jbc.ra120.012601

Figure Lengend Snippet: Figure 9. A synthetic ADAM10/Tspan15 fusion protein is a functional scissor. (A) Schematic representation of the synthetic ADAM10/Tspan15 fusion protein that has the C-terminus of ADAM10

Article Snippet: Mouse ADAM10 tagged at the C-terminus with the C-terminal half of superfolder GFP was generated using a twostep PCR approach in which the GFP tag was at U C L L ibrary Services on M arch 1, 2020 http://w w w .jbc.org/ D ow nloaded from subcloned into pcDNA3.1 mouse ADAM10 (42). pRK5M human ADAM10 was a gift from Rik Derynck (Addgene plasmid # 31717) (43).

Techniques: Functional Assay

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Scatter plot showing cardiac hypertrophy, as measured by Heart weight/Tibia Length (HW/TL) ratio of 8 weeks old 129/Sv mice treated with either vehicle or isoproterenol (ISO) at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 9–10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( B ) Scatter plot representing left ventricular posterior wall thickness of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) Scatter plot indicating the contractile functions of heart as represented by ejection fraction of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( D ) Histogram showing GSK3β activity assay in heart lysates of vehicle or ISO-treated 8 weeks old 129/Sv mice. Mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β peptide substrate, which contains specific phosphorylation residues of GSK3β was measured. n = 10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( E ) Eight weeks old 129/Sv mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy) and the affinity resin immobilized with protein A/G. Western blotting analysis was performed to detect the levels of GSK3β acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as negative control in this assay. Heart tissue lysates (WCL) were probed for indicated proteins by western blotting. ANP was used as a positive control to assess cardiac hypertrophy in ISO infused mice. n = 4 mice per group. # marked western blotting images denotes SIRT2 antibody (#12650; Cell Signaling), used in this assay detects single band. ( F ) Histogram showing relative acetylated GSK3β in vehicle and ISO-treated mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( G ) GSK3β was immunoprecipitated from heart tissues of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology), and the affinity resin with protein A/G immobilized. Western blotting was performed to detect GSK3β interaction with p300 using anti-p300 antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the presence of GSK3β and p300 by western blotting. ( H ) Co-localization of GSK3β with p300 was assessed in 293 T cells by confocal microscopy. The antibodies used are anti-GSK3β (sc-9166, Santacruz), and p300 (05–257, Millipore). DAPI was used to stain the nucleus. Expanded images (right small boxes) show yellow color in the merge image, indicating the co-localization of GSK3β (Green) and p300 (Red) in the nucleus. ( I ) In vitro binding assay to test the direct interaction between GSK3β and p300. Recombinant p300 (Millipore # 2273152) was incubated with recombinant GST or GST-GSK3β, purified from E. coli BL21 (DE3) by affinity chromatography using Glutathione Sepharose 4B. ( J ) Western blotting analysis showing the acetylation and activity of GSK3β in rat neonatal cardiomyocytes infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Depletion of p300 was confirmed by western blotting. GSK3β was immunoprecipitated from control and p300-KD cells using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology) and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β using the anti Ac-Lysine antibody. GSK3β activity was measured by assessing the phosphorylation of glycogen synthase (p–GS). Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cardiomyocyte lysates (WCL). ( K ) Histogram showing the quantification of relative acetylated GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of acetylated GSK3β and GSK3β were quantified by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( L ) Histogram depicting the activity of GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of phospho-glycogen synthase and glycogen synthase were measured by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( M ) Western blotting analysis showing the acetylation of GSK3β in rat neonatal cardiomyocytes infected with either control (Ad-null) or p300 overexpressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting. GSK3β was immunoprecipitated using anti-GSK3β antibody (sc-9166, Santacruz) and the affinity resin with protein A/G immobilized. Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cell lysates (WCL). ( N ) Western blotting analysis showing the activity of GSK3β in rat neonatal cardiomyocytes infected with control (Ad-null) or p300 expressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting and the activity of GSK3β was probed by assessing the levels of p-GS and GS by western blotting. ( O ) Histogram showing the activity of GSK3β in control (Ad-Null) or p300 overexpressing (Ad-p300) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Signal intensities of phospho-glycogen synthase and glycogen synthase were assessed by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( P ) In vitro kinase assay showing the activity of acetylated and non-acetylated GSK3β. Human GSK3β with HA tag was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide. n = 6 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Transfected construct , pcDNA3 Flag HA , Addgene , Plasmid 10792 , 1436 pcDNA3 Flag HA plasmid DNA was a gift from William Sellers.

Techniques: Activity Assay, Immunoprecipitation, Incubation, Phospho-proteomics, Western Blot, Negative Control, Positive Control, Software, Confocal Microscopy, Staining, In Vitro, Binding Assay, Recombinant, Purification, Affinity Chromatography, Infection, Expressing, Luciferase, shRNA, Control, Over Expression, Kinase Assay, Transfection, Plasmid Preparation

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blot analysis of acetylated GSK3β in heart samples of 9 months old WT and SIRT2-KO littermates. GSK3β was immunoprecipitated from heart tissue lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the SIRT2 and GAPDH by western blotting. n = 4 mice per group. ( B ) Histogram showing relative acetylated GSK3β in 9 months old WT and SIRT2-KO mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) GSK3β was immunoprecipitated from heart tissue lysates of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy ), and the affinity resin immobilized with protein A/G. GSK3β interaction with SIRT2 was tested by western blotting using anti-SIRT2 antibody. IgG was used as negative control. Heart lysates was probed for indicated proteins by western blotting. ( D ) In vitro binding assay to test the interaction between GSK3β and SIRT2. Flag-SIRT2 was overexpressed in 293 cells by a plasmid encoding human Flag-SIRT2. Recombinant His or His-GSK3β was purified from E. coli BL21 (DE3) by Ni-NTA affinity chromatography and were incubated with 293 T cell lysates overexpressing human Flag-SIRT2. Interaction between GSK3β and SIRT2 was tested by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( E ) In vitro deacetylation assay showing SIRT2 as GSK3β deacetylase. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The acetylated HA-GSK3β was further incubated with either Flag-tagged SIRT2 or SIRT2-H187Y, which were immunoprecipitated from HEK 293 cell lysates overexpressing respective plasmids encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer. GSK3β acetylation was analyzed by western blotting using anti-Ac-Lysine antibody. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( F ) In vitro kinase assay depicting the activity of acetylated and deacetylated GSK3β. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. Recombinant HA-GSK3β was immunoprecipitated using HA-coupled beads and was acetylated by recombinant p300 in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3β was further deacetylated by either Flag-tagged WT or SIRT2-H187Y (SIRT2-HY), a catalytic inactive mutant of SIRT2, which was immunoprecipitated from HEK 293 cells, overexpressed with plasmid encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer and further enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( G ) Western blot analysis of acetylated GSK3β from control or SIRT2-depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting (control) or siRNA targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by Western blotting. Total cellular acetylation was probed by anti-Ac-Lysine antibody to test the effect of SIRT2 depletion in cardiomyocytes. GSK3β was immunoprecipitated from these cell lysates using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β by anti-Ac-Lysine antibody. Cell lysates (WCL) from control and SIRT2-KD cardiomyocytes were probed for indicated proteins by western blotting. ( H ) Western blotting analysis of hearts lysates from 9 months old WT and SIRT2-KO mice littermates for indicated proteins. n = 4 mice per group. ( I ) Histogram showing activity of GSK3β in WT and SIRT2-KO mice hearts at 9 months of age. GSK3β was immunoprecipitated from the heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β Peptide Substrate, which contains specific phosphorylation residue of GSK3β was measured. n = 6 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( J ) In vitro deacetylation assay to test whether SIRT2 deacetylates K183 residue of GSK3β. HA-tagged GSK3β or GSK3β-K183R was overexpressed in HeLa cells and was immunoprecipitated using HA-coupled beads. HA-tagged WT-GSK3β or GSK3β-K183R were incubated with Flag-SIRT2 immunoprecipitated from HEK 293 T cells using agarose beads conjugated to Anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a deacetylation buffer. Acetylation status of GSK3β was analyzed by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( K ) Histogram showing relative acetylation of HA-tagged GSK3β or GSK3β-K183R, which was incubated with Flag-SIRT2. The data is generated from . Signal intensities of acetylated-GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( L ) Histogram showing binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β. Recombinant His-GSK3β was purified from E. coli BL 21 (DE3) by Ni-NTA affinity chromatography. Purified His-GSK3β was acetylated by recombinant p300 in the presence of Ac-CoA in HAT buffer. Acetylated His-GSK3β was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells. The binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β was assessed by the protocol described in Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( M ) Histogram showing activity of WT or mutants of GSK3β. HA-tagged WT or mutants of GSK3β was immunoprecipitated from HeLa cells transfected with respective plasmids using HA-coupled agarose beads. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Transfected construct , pcDNA3 Flag HA , Addgene , Plasmid 10792 , 1436 pcDNA3 Flag HA plasmid DNA was a gift from William Sellers.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Software, In Vitro, Binding Assay, Plasmid Preparation, Recombinant, Purification, Affinity Chromatography, Incubation, Histone Deacetylase Assay, Transfection, Kinase Assay, Activity Assay, Mutagenesis, Control, Phospho-proteomics, Residue, Generated

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet:

Article Snippet: Transfected construct , pcDNA3 Flag HA , Addgene , Plasmid 10792 , 1436 pcDNA3 Flag HA plasmid DNA was a gift from William Sellers.

Techniques: Knock-Out, Western Blot, Agarose Gel Electrophoresis, Produced, Transfection, Construct, Plasmid Preparation, Modification, Infection, Recombinant, Sequencing, Activity Assay, Mutagenesis, Protease Inhibitor, Microscopy, Software, Membrane, Cell Culture

Journal: Molecular Biology of the Cell

Article Title: The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading

doi: 10.1091/mbc.e10-12-0936

Figure Lengend Snippet: FIGURE 2: GRAF1 is not a general component of focal adhesions but localizes to PLAs. (A) Fluorescence micrograph of HeLa cells coexpressing myc-tagged GRAF1 and GFP-tagged GIT1 and costained for myc and vinculin. Insets show magnifications of the areas indicated by yellow squares. (B) Bar graph showing the percentage of cells in which myc-GRAF1 or myc-GRAF1 R412D was found localized with vinculin in PLAs following the indicated treatments or overexpression of GFP-GIT1. Cells expressing myc-GRAF1 were treated with Y-27632, an inhibitor of rhoA kinase (5 min), or blebbistatin, an inhibitor of nonmuscle myosin II (10 min), and the number of cells where GRAF1 colocalized with vinculin was counted. Bars and error bars correspond to mean and SEM calculated from three independent experiments (n > 250; α = 0.05; two-tailed Fisher’s exact test, ***, p < 0.001). (C) Fluorescence micrograph of cells coexpressing myc-tagged GRAF1 and GFP-cdc42-DA and costained for myc and vinculin. (D) Merged confocal micrograph of a cell expressing src Y527F and GFP-GRAF1 and costained for vinculin. Inset below shows three-dimensional view of the section indicated by the dotted line. Bar graph depicting the percentage of cells with untagged or GFP-tagged GRAF1 localized to vinculin-defined, src-induced podosomes. Bars and error bars correspond to mean and SEM calculated from six independent experiments, each including 30 cells. (E) Fluorescence micrographs of cells treated with the ROCK inhibitor Y-27632 for 15 min or untreated before fixation and staining for vinculin and overexpressed myc-GRAF1. Insets in the top panel show magnification and three-dimensional rotation of the area indicated in the vinculin panel. Arrows indicate basal structures in which GRAF1 and vinculin colocalize. Scale bars: 10 μm.

Article Snippet: Green fluorescent protein (GFP)-tagged cdc42 Q61L (12600; Nalbant et al., 2004), rac1 Q61L (12981), rhoA Q63L (12968; Subauste et al., 2000), and untagged src Y527F (13660) were purchased from Addgene (Cambridge, MA).

Techniques: Fluorescence, Over Expression, Expressing, Two Tailed Test, Staining

Journal: Molecular Biology of the Cell

Article Title: The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading

doi: 10.1091/mbc.e10-12-0936

Figure Lengend Snippet: FIGURE 4: Membrane remodeling by GRAF1 and uptake of CTxB are stimulated at the leading edge of migrating cells. (A) Fluorescence micrographs of myc-GRAF1–expressing HeLa cells incubated with CTxB-Alexa555 for 5 min before washing, fixation, and costaining for vinculin. (B) Confluent wild-type MEF monolayers were wounded by scratching, and cells were allowed to migrate into the wound for 4–6 h. CTxB-555 and Tfn-647 were then added to migrating cells for 2 min of uptake at 37°C. Cells were acid-stripped and fixed and were then labeled for endogenous paxillin. Arrows indicate colocalization between paxillin and CTxB but not Tfn. (C) Twenty-four cells across three independent experiments were treated as in (B), and the percentage of paxillin (green) pixels that colocalized with either CTxB (red) or Tfn (blue) pixels was calculated using Volocity version 3.0. Bars and error bars correspond to mean and SEM (n = 12–15; α = 0.05; Student’s t test, **, p < 0.01). (D) Fluorescence micrographs of HeLa cells expressing myc-GRAF1 and DA rac1 (GFP-Rac-DA; left panels) and incubated with CTxB- Alexa555 for 5 min (right panels). The length of GRAF1 tubules was measured in fluorescence micrographs of seven different cells (n = 193) as described. The mean length is indicated by a red line and the error bars represent standard deviation (SD) above and below the mean. (E) Fluorescence micrographs of cells expressing myc-GRAF1 and DA rhoA (GFP-rhoA-DA) and costained for vinculin. GRAF1-positive tubules were found in 14.3 ± 3.7%, as determined from three independent experiments (n = 124; error values represent SEM). Length of GRAF1 tubules in μm was measured from nine different cells (n = 108) and depicted as in Figure 4D. Scale bars: 10 μm.

Article Snippet: Green fluorescent protein (GFP)-tagged cdc42 Q61L (12600; Nalbant et al., 2004), rac1 Q61L (12981), rhoA Q63L (12968; Subauste et al., 2000), and untagged src Y527F (13660) were purchased from Addgene (Cambridge, MA).

Techniques: Membrane, Fluorescence, Expressing, Incubation, Labeling, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 1. Generation of Nckx4/ knock-out mice. A, diagram illustrating the Nckx4 (slc24a4) locus, the targeting vector, and the targeted locus. The location of Nckx4 exons 5, 6, 7, and 8 is indicated. The neomycin (neo) gene driven by a pol II promoter replaced exons 6 and 7 in the targeted locus. The thymidine kinase gene driven by a herpes simplex virus promoter was used for negative selection. The location of the probe used for Southern analysis and the length of the two diagnostic NsiI fragments of 9.9 and 4.9 kb are illustrated at the top and bottom of the diagram. The position of the primers used for PCR analysis, P1, P2, and P3, which generate diagnostic fragments of 175or350bparealsoindicated.B,genotypeanalysisbySouthernblot(left)or PCR (right) of DNA from wild-type (/), heterozygous (/), or Nckx4 knock-out(/)animals.C,Northernblot(leftpanel)analysisof10goftotal RNA samples, or immunoblot (right panel) analysis of 20 g of membrane proteins samples, isolated from the indicated tissues of mice of different genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Plasmid Preparation, Virus, Selection, Diagnostic Assay, Western Blot, Membrane, Isolation

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 2. Nckx4 knock-out does not change brain morphology or the expression of other Na/Ca2-exchangers. A, serial coronal sections through the hypothalamus of wild-type adult mice were labeled for Nckx4 transcripts with an antisense or a sense control probe, as indicated. B, hematoxylin and eosin-stained coronal paraffin sections through the PVN (wild-type, /; Nckx4 knock-out, /). No differences were observed between genotypes in this or in other brain regions examined in either coronal or parasagittal stained sections (data not shown). In both panels A and B, the right-hand images are magnified views of the area in the box in the corresponding left-hand images. Scale bars: left-hand images, 1 mm; right-hand images, 200 m. C, Northern blots of 10 g of total RNA samples isolated from adult brain of wild-type (/), heterozygous (/), or Nckx4 knock-out (/) mice were analyzed with probes for the indicated Na/Ca2-exchangers genes. The blots were then stripped and reprobed for Gapdh as a loading control. All images in this figure are representative of independent experiments from 3 different animals of each genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Expressing, Labeling, Control, Staining, Northern Blot, Isolation

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 3. Nckx4/ mice are anorexic and hypophagic. Knock-out (/) and wild-type (/) and heterozygous (/) littermates, as indicated, were fed ad libitum either standard or enriched laboratory chow (4.5% or 9% fat). A, animals were weighed weekly from 4 weeks of age onwards (n 12–15 for each group; however, measurements were not made for all animals for all time points, all conditions or both sexes; therefore, some data points corre- spond to a smaller n value). B, animals were weighed at birth (P0; n 10) and then at weaning (n 63, 107, and 56 for wild-type, heterozygous, and knock- out, respectively). C, weight (n 8) and length (nose to anus; n 10) were measured on adult animals. D, food (n 8 for females and n 10 for males) and water consumption (n 6) were measured in adult animals. In all cases the mean S.E. is plotted. Statistical significance of differences between knock-out and wild-type is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 4. Body temperature and tissue weight. A, basal body temperature was determined rectally in free-fed or fasted adult animals (n 4–8). B, tissues were extracted from adult (5–6 months of age) animals (n 5–7), blotted dry and weighed. Fat corresponds to total abdominal cavity adipose tissue. Average values S.E. are plotted. Statistical significance of the differences between wild-type (/; WT) and Nckx4 knock-out (/; KO) is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. C, microscopic images of hematoxylin and eosin-labeled sections from paraffin-embedded samples of abdominal fat pad or liver are shown. Scale bar, 100 m. These images are representative of samples from at last 5 pairs of animals of each genotype.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Labeling

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 5. Health parameters, motivation and olfaction in Nckx4/ knock-out and wild-type (/) mice. A, a glucose tolerance test was performed on 5–6-month-old animals following overnight fasting. Glucose (2 mg/g body weight) was injected intraperitoneally, and blood glucose was measured just prior and at intervals thereafter. Average values S.E. are plotted (n 6). B, a Kaplan Meier survival curve is plotted for 18 wild-type (/) and 13 Nckx4 knock-out (/) animals. A log-rank (Mantel-Cox) test indicates a significant difference between genotypes, p 0.05. C, cumulative food consumption was measured in adult mice immediately following a 24-h fasting period (n 11). D, time taken to find buried urine-scented litter (left) or buried food pellet (right) was measured for adult mice (n 11–13). Statistical significance of the differences between genotypes determined by analysis of variance using the Bonferroni post-test is indicated where present: *, p 0.05; ***, p 0.001.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Injection

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 7. Neurons in the PVN are activated in Nckx4/ mice. c-Fos expression was detected by immunofluorescence in sections from hypothal- amus of adult mice. WT, wild-type; KO, Nckx4/. All animals were fasted for 48 h and subsequently treated for 30 min prior to sacrifice either with glucose injection (intraperitoneally; 2 mg/g body weight) or by perfusion with Ca2- free Krebs-Henseleit solution containing 1 mM EGTA, normal Krebs-Henseleit solution containing 100 nM insulin, or normal Krebs-Henseleit solution con- taining 1 M of the MC4R antagonist, SHU9119 (SHU). The total number of c-Fos positive nuclei contained within the PVN area were counted for each section, and the averaged data S.E. from 4 to 7 independent experiments are shown in the panel at the lower right. Statistical significance is indicated where present: compared with wild-type untreated: *, p 0.05; **, p 0.01; ***, p 0.001; compared with glucose-treated wild-type: #, p 0.05; com- pared with knock-out untreated: †††, p 0.001. Scale bar, 500 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Expressing, Immunofluorescence, Injection, Knock-Out

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 6. Blood hormones and hypothalamic mediators. A, serum sam- ples from overnight fasted female (F) or male (M) animals of wild-type (/) or Nckx4 knock-out (/) genotype (n 7–10) were tested for the indicated factors. Levels are plotted normalized to the wild-type female levels in each case. Statistical significance of differences between knock-out and wild-type for each gender is indicated where present: *, p 0.05; **, p 0.01; ***, p 0.001. The values corresponding to 100% are: insulin, 1.51 ng/ml; leptin, 55.5 ng/ml; glucose, 261 mg/dl; triglycerides, 0.49 mg/ml; T4, 5.0 g/dl; cortisol, 4.1 g/dl; MSH, 110 pmol/liter. B, localization of MSH and AgRP was deter- mined by immunofluorescence in coronal sections through the hypotha- lamic region of adult female wild-type (/) or Nckx4 knock-out (/) mice. For illustrative purposes, bilaterally symmetric image halves are compared between Nckx4/ and Nckx4/ animals for sections taken at the level of the PVN and ARC. The PVN and ARC regions are circled. Scale bar, 500 m. The right-hand most image pairs are magnified views of the ARC regions from the central image pairs, illustrating the punctate nature of staining, consis- tent with packaging of peptide hormones in vesicles destined for secretion. Scale bar, 100 m. These images are representative of three independent experiments.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 9. MSH-induced Ca2 signals. A, the fura-2 340/380 fluorescent exci- tation ratios are shown for individual neurons, each plotted with a uniquely colored trace, from primary hypothalamic cultures from either wild-type (WT) or Nckx4 knock-out (KO) mice. Cells were continually perfused with Hepes- buffered saline, containing either 1 M MSH or 75 mM KCl where indicated. B, the number of cells in each microscopic field that responded with a change in fura-2 ratio, as shown in panel A, following MSH treatment is plotted. Aver- ages S.E. for 18–20 microscopic fields from six independent cultures are shown. ***, p 0.001. C, GT1–7 cells were infected with lentivirus expressing mouse NCKX4, or a deleted control construct, and subsequently assayed for Ca2 response 2 days later. The fura-2 340/380 excitation ratios for individual responding cells are shown, each plotted with a uniquely colored trace. D, the average number of responding cells per field is summarized. n 9; **, p 0.01. E, GT1–7 cells in Opti-MEM were treated with vehicle only (control) or with 10 M SKF96365 or 10 M U73122 at 37 °C for 15 min with the indicated concentration of MSH, followed by extraction and determination of cAMP levels (n 5–8).

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Knock-Out, Saline, Infection, Expressing, Control, Construct, Concentration Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 8. The Nckx4/ phenotype is MC4R-dependent. Age-matched adult mice of different genotypes were compared: WT, wild-type; N4KO, Nckx4/; M4KO, Mc4r/; DKO, Nckx4/; Mc4r/. A, body weight (n 6–9); B, food consumption (n 10); C and D, c-Fos immunofluorescence in coronal hypothalamic sections of fasted animals, as described in the legend toFig.7(n4–7).InpanelsAandB,N4KO,M4KO,andDKOareallsignificantly different from WT, and in panels A, B, and D, M4KO and DKO are significantly different from N4KO, with p 0.001 in all cases. Scale bar, 500 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 11. Control of oxytocin expression in the hypothalamic paraven- tricular nucleus. Oxytocin expression was detected by immunofluorescence in adult mice. WT, wild-type; KO, Nckx4/. Animals were fasted for 48 h and then subjected to a 30-min transcardial perfusion with Krebs-Henseleit solu- tion containing 1 M MSH, 10 M SKF96365, or 10 M U73122, as indicated. The oxytocin-positive cells were scored as being of high or low staining inten- sity, and the numbers pooled across all sections from each of five animals per treatmentgroup.Thetotalnumberofcellsscoredvariedfrom522to1288per animal, but the averaged values were not different between groups (800 cells). The averaged ratios of high/low stained cells S.E. are shown in the panel at lower right. Statistical significance is indicated where present: com- pared with wild-type untreated: **, p 0.01; ***, p 0.001. Scale bar, 100 m.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Control, Expressing, Immunofluorescence, Staining

Journal: Journal of Biological Chemistry

Article Title: An Essential Role for the K+-dependent Na+/Ca2+-exchanger, NCKX4, in Melanocortin-4-receptor-dependent Satiety

doi: 10.1074/jbc.m114.564450

Figure Lengend Snippet: FIGURE 12. A model for MC4R-dependent Ca2 signaling and the role of NCKX4. MSH binding to the MC4R is shown to active Gq, via the exchange of GTP for GDP (upper row). It is important to note that MC4R may constitutively activate Gq even in the absence of ligand (54). So activated, Gq goes on to bind and stimulate PLC-, which subsequently cleaves phosphatidylinositol 1,4-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The membrane-bound diacylglycerol then activates the Ca2-selective entry channel, TRPC6, which results in an elevation of cytosolic [Ca2] and consequent neuronal activation, which may include the initiation of action potentials (15) and the release of oxytocin (16, 41) (lower row). Normally, NCKX4 acts locally to extrude the Ca2 that enters via TRPC6, thus moderating the signaling pathway so only strong signals drive neuronal activation. In the absence of this moderating influence, even weak, constitutive, signaling via MC4R can result in sufficient Ca2 entry to initiate neuronal activation.

Article Snippet: A fragment comprising the CaMKIIa promoter from the pLentiCamKII-ChETA-EYFP (a kind gift of Dr. Karl Deisseroth obtained as Addgene plasmid 26967) (28) was inserted at the 5 end of the Nckx4 cDNA.

Techniques: Binding Assay, Membrane, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 1. Keap1 and Nrf2 expression in RANKL-stimulated RAW 264.7 cells. The expressions of Keap1 and Nrf2 and the Nrf2/Keap1 ratio were examined by real time PCR at day 1 (A) and day 2 (B). Gene expression was calibrated using the RPS18 housekeeping gene, and values indicating the fold-change from controlareshown.Openbarsrepresentcontrols,andclosedbarsrepresentstimulationbyRANKL.CshowsnucleartranslocationofNrf2inRAW264.7cells.Equal amounts of nuclear protein extracted from control or RANKL-stimulated RAW 264.7 cells were electrophoresed and transferred to PVDF membrane. The membrane was subjected to Western blot analysis for Nrf2 and histone H3. Densitometry analysis was used to calculate the ratio relative to the control, and the mean values of three experiments are shown. The data shown are representative of three independent experiments performed in triplicate. *, p 0.05 versus control.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Control, Membrane, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 3. Nuclear translocation of Nrf2 is increased by Nrf2 overexpression and decreased by Keap1 overexpression or Nrf2 knockdown, altering the transcription of cytoprotective enzymes. The expressions of Nrf2 (A) and Keap1 (B) were examined by real time PCR at day 1. Gene expression was calibrated using the RPS18 housekeeping gene, and values indicating the fold-change from control are shown. A, compares expression levels for human Nrf2 between control (cont), transfection with human Nrf2 (up-regulation), and transfection with Nrf2 RNAi (knockdown); B, compares expression levels for human Keap1 between control and transfection with human Keap1 (up-regulation). The data represent the means of three independent experiments performed in triplicate. *, p 0.05 versus control. C, nuclear translocation of Nrf2. Equal amounts of nuclear protein were electrophoresed and transferred to PVDF membrane. The membrane was subjected to Western blot analysis for Nrf2 and histone H3. The upper panels show the comparison between mock and Nrf2 transfection, and the lower panels show the comparison between nonsilencing and Nrf2 siRNA. Densitometry analysis was used to calculate the ratio relative to the control, and the mean values of three experiments are shown for Nrf2, above each respective panel. NS, no significant difference between groups.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Translocation Assay, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Control, Expressing, Transfection, Membrane, Western Blot, Comparison

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 5. Detection of intracellular ROS. Control RAW cells and cells with overexpression/knockdown of Keap1/Nrf2 were stimulated with sRANKL for 6 h, and the ROS levels were examined using flow cytometry. A, RANKL induced intracellular ROS. The red line indicates the control, and the cyan line indicates stimulation with RANKL. The data shown are representative of three independent experiments performed in triplicate. B, Nrf2 knockdown increased intracel- lularROSlevelsinRANKL-stimulatedRAWcells.TheredlineindicatesRANKLstimulationofcellstransfectedwithnonsilencingsiRNA,andthecyanlineindicates RANKL stimulation of cells transfected with Nrf2 siRNA. The data shown are representative of three independent experiments performed in triplicate. C, intra- cellularROSlevelsinRANKL-stimulatedRAWcellsweredecreasedbyNrf2overexpressionandincreasedbyKeap1overexpression.TheredlineindicatesRANKL stimulation of cells transfected with mock vector; the cyan line indicates RANKL stimulation of cells transfected with the Keap1 expression plasmid, and the orange line indicates RANKL stimulation of cells transfected with the Nrf2 expression plasmid. The data shown are representative of three independent experiments performed in triplicate.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Control, Over Expression, Knockdown, Flow Cytometry, Transfection, Plasmid Preparation, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 4. Expression of cytoprotective genes was up-regulated by Nrf2 overexpression and down-regulated by Keap1 overexpression or Nrf2 knockdown. The expressions of HO-1, NQO1, and GCS were examined by real time PCR at day 2 (A). Gene expression was calibrated using the Rps18 housekeeping gene, and values indicating the fold-change from control are shown. The data represent the means of three independent experiments per- formedintriplicate.*,p0.05versuscontrol.B,WesternblotanalysisforHO-1,NQO1,andGCS.Cytoplasmicextractscontainingequalamountsofproteinwere electrophoresed and transferred to PVDF membrane. The membrane was subjected to Western blot analysis. To confirm the equivalence of loaded cytoplas- micprotein,TGXStain-FreePrecastGelswereused.ProteinsintheelectrophoresedgelwerevisualizedwithUVtreatmentbeforetransfertoaPVDFmembrane. Visualized protein on the membrane was imaged under UV transillumination, and the value of the total band density was used for calibration. Densitometry analysis was used to calculate the ratio relative to the control, and the mean values of three experiments are shown above each panel. NS, no significant difference between groups.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Expressing, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Gene Expression, Control, Membrane, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 6. Osteoclastogenesis assay. A, mock, Nrf2, and Keap1 expression plasmids were transfected into RAW 264.7 cells; the cells were then stimulated with RANKL for 3 days, and TRAP staining was performed. The data shown are representative of three independent experiments performed in triplicate. *, p 0.05 versus mock transfection. Bar, 100 m. Arrowheads indicate TRAP multinucleated cells. B, nonsilencing or Nrf2 siRNA was transfected into RAW 264.7 cells; the cells were then stimulated with RANKL for 3 days, and TRAP staining was performed. The data shown are representative of three independent experiments performed in triplicate. *, p 0.05 versus nonsilencing siRNA transfection. Bar, 100 m. Arrowheads indicate TRAP multinucleated cells. C, osteoclastogenesis assay using mouse primary peritoneal macrophages. The cells were transfected with the Nrf2 or Keap1 expression plasmid, or Nrf2 siRNA, 12 h before stimulation with RANKL. The cells were stimulated with RANKL for 7 days, and TRAP staining was performed. The culture medium was exchanged once at day 4. All culture media for peritoneal macrophage culture were supplemented with recombinant macrophage colony-stimulating factor (20 ng/ml). The data shown are representative of three independent experiments performed in triplicate. Bar, 100 m. *, p 0.05 versus stimulation with RANKL alone. NS, no significant difference between groups.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Expressing, Transfection, Staining, Plasmid Preparation, Recombinant

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 8. Resorption assay. Mouse primary peritoneal macrophages were performed Nrf2 transfection or RNAi knockdown and stimulated with RANKL. The photographs shown are representative of three experiments (A). Arrowheads indicate resorbed lacunae on substrate. Bar, 100 m. Mean percent resorbed area was calculated from randomly selected six images using ImageJ software (B). *, p 0.05 versus stimulation with RANKL.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Transfection, Knockdown, Software

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 9. Local Nrf2 overexpression ameliorates bone destruction in vivo. LPS (10 g/site) or PBS were injected five times into each mouse, every other day, with or without local human Nrf2 gene transfer. A, real time PCR analysis was performed using three RNA samples per group. Mean Ct values of human Nrf2 in each group are given under “Results.” *, p 0.05 between groups. B, x-ray microtomographic images were taken using five samples per group, and representative photographs are shown. Arrowheads indicate resorbed lacunae or holes formed in the cranial bone. Bar, 1 mm. C, resorbed area in the cranial bone was calculated using ImageJ software. Mean values are shown for percent resorbed (calculated from the ratio of the number of pixels in the resorbed area in the cranial bone to the number of pixels in the cranial bone in the analyzed image). *, p 0.05 versus control. NS, no significant difference versus control. †, p 0.05 between groups.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Over Expression, In Vivo, Injection, Real-time Polymerase Chain Reaction, Software, Control

Journal: Journal of Biological Chemistry

Article Title: The Keap1/Nrf2 Protein Axis Plays a Role in Osteoclast Differentiation by Regulating Intracellular Reactive Oxygen Species Signaling

doi: 10.1074/jbc.m113.478545

Figure Lengend Snippet: FIGURE 10. Schematic illustration of the proposed mechanism by which the Keap1/Nrf2 axis regulates osteoclastogenesis. First, RANKL signaling attenuates the Nrf2/Keap1 ratio, which weakens the transcription of Nrf2-de- pendent cytoprotective enzymes. Second, diminished levels of cytoprotec- tive enzymes cause augmented intracellular ROS levels. Finally, osteoclasto- genesis and osteoclast activation are induced.

Article Snippet: Overexpression of Keap1 and Nrf2—Expression plasmids encodinghumanKEAP1(hrGFP-Keap1;Addgeneplasmid28025) (24) and human NRF2 (pcDNA3-EGFP-C4-Nrf2; Addgene plasmid 21549) (25)were obtained, respectively, fromDr.QingZhong (University of California, Berkeley, CA) and Dr. Yue Xiong (University of North Carolina, Chapel Hill) via Addgene.

Techniques: Activation Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Schematic of Tspan12 purification. Human Tspan12∆C (truncated at residue 252 after the fourth transmembrane domain) was tagged with the Rho1D4 tag TETSQVAPA and expressed in Sf9 cells by baculovirus. Detergent-solubilized Tspan12 was affinity purified on anti-Rho1D4 antibody resin followed by size exclusion chromatography. Panel A was created with BioRender.com . ( B ) Superose 6 10/300 size exclusion traces of Tspan12 purified in DDM (teal) or exchanged into glyco-diosgenin (GDN) during the affinity chromatography step (pink); the final concentrated product in GDN showed no signs of aggregation (purple). Indicated fractions of GDN-solubilized Tspan12 (pink trace) were run on ( C ) SDS-PAGE; boxed fractions were pooled and concentrated. ( D ) Size exclusion traces of empty nanodiscs (black) or Tspan12 reconstituted into excess MSP1D1 nanodiscs (pink). Tspan12 content of fractions was quantified by dot blot (anti-Rho1D4) and dot intensity was plotted accordingly (green). ( E ) Peak Tspan12-containing fractions from ( D ) were affinity purified on anti-Rho1D4 resin. Shown is an SDS-PAGE gel of the load, flowthrough, and eluate. ( F ) Uranyl acetate negative stain micrograph of final nanodisc-reconstituted Tspan12. Scale bar is 20 nm. Figure 1—figure supplement 1—source data 1. Original files of gels in . Figure 1—figure supplement 1—source data 2. Labeled gels in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Purification, Residue, Affinity Purification, Size-exclusion Chromatography, Affinity Chromatography, SDS Page, Dot Blot, Staining, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Schematic of biolayer interferometry (BLI) setup for Tspan12-Norrin binding: Tspan12 lacking the C-terminal tail (∆C), inserted into biotinylated nanodiscs, is immobilized on a streptavidin-coated biosensor, and Norrin association and dissociation are monitored in real time. ( B ) BLI traces of Norrin at indicated concentrations binding to and dissociating from Tspan12. ( C ) Steady-state binding curve fit to Norrin-Tspan12 binding (mean ± SD from three independent replicates at each concentration of Norrin) gives a K D of 10.4±1.2 nM (mean ± SEM). ( D ) Observed association rate constant (K obs ), determined from fitting BLI association traces (mean ± SD in three independent experiments), is linearly dependent on Norrin concentration with a slope K on = 0.00019 ± 0.00003 nM –1 s –1 (mean ± SEM). When combined with the K off = 0.0014 ± 0.00016 s –1 (mean ± SEM) determined from fitting the dissociation traces, we obtain a kinetic K D of 7.4±1.4 nM (mean ± SEM). ( E ) BLI traces of the soluble MBP-tagged Tspan12 LEL domain, at the indicated concentrations, associating to and dissociating from a biosensor loaded with MBP-tagged Norrin. Kinetic fitting gives an apparent affinity of 16±3 nM (mean ± SEM). ( F ) BLI traces of 10, 32, or 100 nM Norrin show no binding to a biosensor loaded with a nanodisc-embedded chimeric Tspan12 with the LEL replaced by that of Tspan11. Figure 1—source data 1. Steady-state interference shift and K obs values used to generate .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Concentration Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Biolayer interferometry (BLI) traces showing non-biotinylated Tspan12 nanodiscs in solution binding to biosensor-immobilized biotinylated MBP-Norrin (flipped setup compared to ). Overlaid fits (black dashed line) give an apparent affinity of 34±11 nM. ( B ) BLI traces showing weak binding of indicated concentrations of Tspan12 in glyco-diosgenin (GDN) detergent to biosensor-immobilized biotinylated MBP-Norrin. ( C ) BLI trace showing no binding of MBP-tagged Tspan12 large extracellular loop (LEL) at 18 µM to biosensors loaded with nanodisc-embedded Fzd4. ( D ) BLI traces showing no binding of the soluble Fzd4 CRDL up to 32 µM to biosensors loaded with nanodisc-embedded Tspan12.

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Cartoon of tetraspanin structure, comprised of transmembrane helices 1–4, a small extracellular loop (SEL, between helices 1 and 2) and a large extracellular loop (LEL, between helices 3 and 4). Eye icon indicates the viewing angle of AlphaFold models in the remainder of this figure ( B, D, F, I–P ) relative to this cartoon. ( B ) The structure of full-length human Tspan12 alone was predicted with AlphaFold. The best-scoring model is shown, colored by the per-residue predicted local distance difference test (pLDDT) confidence metric. ( C ) The predicted aligned error (PAE) for the model in B. The position of the LEL (darker purple) appears as a darker blue square in the heat map with low PAE (yellow box). The position of C-terminal residues (‘CT‘, red) is indicated; the heat map shows high PAE values for the C-terminus relative to the rest of the protein, indicating poor prediction of the relative positioning of the C-terminus. ( D ) The structure of full-length human Tspan12 together with one Norrin protomer (residues 25–133) was predicted with AlphaFold-Multimer and the best-scoring model is shown, colored by pLDDT. ( E ) The predicted aligned error for the model shown in D. The position of Norrin, Tspan12, and the LEL along the axes are indicated. Note low PAE between the LEL and parts of Norrin (yellow boxes). ( F ) The structure of full-length human Tspan12 together with two copies of Norrin (residues 25–133) was predicted with AlphaFold-Multimer and the best-scoring model is shown, colored by pLDDT. ( G ) The predicted aligned error for the model shown in F. The position of both copies of Norrin, Tspan12, and the LEL along the axes are indicated. Note low PAE between the LEL and parts of both Norrin protomers (yellow boxes). ( H ) Top: The structure of Norrin within the predicted structure of Tspan12+1 Norrin protomer (yellow), and within the predicted structure of Tspan12+2 Norrin protomers (orange), matches the crystal structure of Norrin (5BQ8 chains A and B; gray) with RMSDs of 0.383 and 0.413 Å respectively. Below: β strands 1, 2, 3, 4, 5, and 6 of one Norrin protomer, colored red, orange, yellow, green, blue, and purple, respectively; strands 5 and 6 are predicted to comprise the Tspan12 binding site. ( I ) The predicted model of Tspan12 alone (light blue) is similar to the predicted models with one (purple/yellow) or two (pink/orange) copies of Norrin, which each align with RMSDs of 1.247 and 1.057 Å, respectively, to the model of Tspan12 alone. Aligning only the LELs as shown gives RMSDs of 0.293 and 0.341 Å, respectively, and illustrates the slight variation in the predicted angle between the TMs and the LEL. The predicted position and orientation of Norrin relative to the LEL is unchanged between the one-protomer and two-protomer models. ( J ) Tetraspanin LELs are composed of helices A, B, C, D, and E. Helices C and D are the least conserved and are implicated in binding partner interactions . Black double lines represent disulfide bridges. Viewing angle is indicated by the eye in A. ( K ) Close-up of aligned LELs from I. ( L ) The predicted structure of the Tspan12 LEL (from the 1 Tspan12 : 1 Norrin protomer model; residues 123–215; purple) aligns to the experimentally determined structure of CD81 LEL (5TCX residues 123–198; gray), with an RMSD of 3.997 Å. CD81 is a C4 tetraspanin; disulfide bonds for Tspan12 and CD81 are shown in pink and black, respectively. ( M ) The predicted Tspan12 LEL (purple) aligns to the Uroplakin 1A LEL (8JJ5, residues 125–226; gray), a C6 tetraspanin, with an RMSD of 4.215 Å. ( N ) The predicted Tspan12 LEL (purple) aligns to the Peripherin-2 LEL (7ZW1, residues 126–258; gray), a C6 tetraspanin, with an RMSD of 2.528 Å. ( O ) The predicted interaction between Norrin and Tspan12 involves helices C and D of the LEL and residues on β5 and β6 of Norrin. ( P ) The predicted interaction between Norrin and Tspan12 colored by surface electrostatics (APBS) shows a highly polar interaction involving a basic patch on Norrin and an acidic patch on Tspan12.

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Residue, Binding Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) AlphaFold model of one Norrin protomer (yellow) bound to Tspan12 (purple), with the expected location of the plasma membrane shown in gray. ( B ) Zoomed view of the predicted Tspan12/Norrin binding site, front and rear view (flipped 180°). Predicted polar interactions are indicated with dark gray dashed lines. Within the binding interface, Site 1 (red), Site 2 (teal), and Site 3 (blue) are indicated. Bold residue labels indicate residues mutated for binding assays. ( C ) AlphaFold model of Tspan12 bound to Norrin dimer and ( D ) zoomed view of indicated area, showing the predicted polar interaction between residue S82 on the second Norrin protomer (orange) and residue E170 on Tspan12, termed Site 4 (orange). ( E ) Binding affinities (mean ± SD) for the indicated Norrin mutants binding to full-length wild-type (WT) Tspan12 and ( F ) WT Norrin binding to the indicated Tspan12 mutants, calculated from association and dissociation fits to biolayer interferometry (BLI) traces of 32 nM Norrin binding to Tspan12 in triplicate (see ). Colors correspond to sites within the binding interface. Kinetic traces and kinetic constants are shown in , and affinities and kinetic constants are reported in . Figure 2—source data 1. Norrin-Tspan12 mutant affinities used to generate .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Residue, Mutagenesis

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Stain-free non-reducing SDS-PAGE gels of Sf9 supernatant media (‘load’), amylose resin flowthrough (‘flothru’), amylose eluate (‘elu’), and pooled fractions after size exclusion (‘SEC’) for wild-type (WT) and indicated mutant MBP-Norrin. ( B ) Stain-free SDS-PAGE gels of DDM-solubilized Expi293 membranes (‘sol’), supernatant post-ultracentrifugation (‘load’), 1D4 antibody resin eluate (‘elu’), and pooled fractions after size exclusion (‘SEC’) for full-length WT and indicated mutant Tspan12. Figure 2—figure supplement 2—source data 1. Original files of gels in . Figure 2—figure supplement 2—source data 2. Labeled gels in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Staining, SDS Page, Mutagenesis, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative biolayer interferometry (BLI) association and dissociation traces of 32 nM wild-type (WT) or mutant Norrin binding to immobilized WT Tspan12, and for ( B ) 32 nM WT Norrin binding to immobilized WT or mutant Tspan12. Colors correspond to sites within the binding interface defined in . ( C and D ) Rate constant K on (mean ± SD) calculated from association and dissociation fits to traces shown in A and B, respectively; each mutant was measured in triplicate. ( E and F ) Rate constant K off (mean ± SD) calculated from dissociation fits to traces shown in A and B, respectively; each mutant was measured in triplicate. Across the board, differences in K D for mutants compared to WT were driven largely by differences in K off . Kinetic constants reported in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Mutagenesis, Binding Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative biolayer interferometry (BLI) association and dissociation traces showing 32 nM mutant Norrin binding to immobilized mutant Tspan12 relative to the WT/WT binding (gray) for mutants at Site 1 (red), Site 2 (teal), Site 3 (blue), and Site 4 (orange). For each site, mutated residues on Norrin and Tspan12 are predicted to interact according to the AlphaFold structure; in the case of charge-swapped mutations (i.e., Sites 1, 3, and 4), we hypothesized that the mutations to the two proteins may be compensatory. ( B ) Binding affinities calculated from kinetic fits to traces in A for the indicated mutant pairs. The charge-swapped mutants at Site 1, Norrin R107E/R115E and Tspan12 E173K/D175K, do not bind appreciably at 32 nM Norrin. At Site 3, charge-swapped Norrin K102E/R121E and Tspan12 196K/S199K binding is rescued to WT/WT levels compared to the much weaker binding affinities of either mutant for its WT counterpart: i.e., the mutations are compensatory. At Site 4, the Norrin S82D mutation partially rescues the deleterious effects of Tspan12 E170K. Data represent mean ± SD from three replicates. Binding affinities and kinetic constants reported in . ( C ) K on and ( D ) K off , calculated from each individual trace shown in A, measured in triplicate. Plots represent three replicates ± SD, and kinetic constants are reported in . WT, wild-type.

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Mutagenesis, Binding Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) The AlphaFold-predicted structure of Tspan12 (purple) bound to a Norrin dimer (yellow/orange), aligned to the crystal structure (5BQC) of a Norrin dimer bound to the Fzd4 CRD (blue) and sucrose octasulfate (SOS) (red). A zoomed view of the indicated region shows an overlap in the predicted binding site of Tspan12 with that of SOS, suggesting that Tspan12 and SOS cannot bind simultaneously to Norrin. ( B ) Displacement of Norrin (32 nM) from immobilized Tspan12 by increasing concentrations of SOS, as measured by biolayer interferometry (BLI) (see ). The equilibrium binding signal is plotted as a percent of signal in the absence of SOS (mean ± SD of three independent experiments), yielding a K i of 34±4 µM. ( C ) Side view of structures in A. A zoomed view of the indicated region shows that Tspan12 is predicted to occupy a site on Norrin adjacent to, but not overlapping with, the Fzd4 binding site; adjacent residues are shown. Norrin from the Tspan12-bound AlphaFold model (yellow) and the Fzd4 CRD-bound crystal structure (5BQC, gray) are overlaid. ( D ) Fzd4 CRDL does not fully compete with Tspan12-Norrin binding, as shown by equilibrium binding of 32 nM Norrin to Tspan12 immobilized on paramagnetic particles in the presence of increasing concentrations of purified Fzd4 CRDL. Bound Norrin and Norrin in the supernatant were both quantified by western blot (anti-Rho1D4; see ) and used to calculate bound Norrin as a percentage of total Norrin. The expected competition curve, assuming fully competitive binding sites, was simulated (gray dashed line) given starting concentrations of 50 nM Tspan12 and 32 nM Norrin, and binding affinities of 10.4 nM for Tspan12-Norrin and 200 nM for Fzd4 CRDL-Norrin. However, the data better fit a model in which CRDL binding to Norrin shifts Norrin affinity for Tspan12 (blue line). Data represent mean ± SD of three replicates. ( E ) BLI traces of a ternary Fzd4-Norrin-Tspan12 large extracellular loop (LEL) complex. Biosensors loaded with nanodisc-embedded Fzd4 were first saturated with 100 nM Norrin, then bound to 32, 100, or 320 nM Tspan12 LEL. ( F ) BLI traces of ternary complex formation. Biosensors loaded with maltose binding protein (MBP)-tagged Norrin were pre-incubated in buffer or saturated with Fzd4 CRDL (5 µM), then bound to 100 nM MBP-tagged Tspan12 LEL (±5 µM CRDL). Tspan12 LEL did bind to Norrin in the presence of the Fzd4 CRDL (dark purple; apparent K D = 27 ± 2.8 nM), albeit more weakly than it bound to Norrin alone (light purple; apparent K D = 16 ± 1.8 nM; see also ). Binding affinities were obtained from kinetic fits (black dotted line) to association and dissociation traces of MBP-LEL (100 nM) from three independent experiments. Figure 3—source data 1. Interference shift and band quantification values used to generate .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Purification, Western Blot, Incubation

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative biolayer interferometry (BLI) traces of 32 nM Norrin binding to biosensor-immobilized Tspan12 in the presence of increasing concentrations of SOS. ( B ) Representative western blot of Norrin in the supernatant ( S ) or remaining bound ( B ) to Tspan12 or empty nanodiscs immobilized on paramagnetic particles in the presence of 0, 0.1, 0.4, 1.6, 6, 25, or 100 µM of purified Fzd4 CRD-linker domain, with both Norrin and Tspan12 detected by anti-Rho1D4 antibody. Norrin is detected within the dynamic range, as shown by concentration standards loaded at left. Tspan12 forms SDS-induced dimers, which can be detected at about 50 kDa. ( C ) BLI traces showing binding of the Fzd4 CRDL to biosensor-immobilized MBP-Norrin, which reaches saturation at 3.2 µM CRDL. Equilibrium binding values obtained from the overlaid fits (black dashed lines) give a steady-state affinity of 122±38 nM (mean ± SEM), in agreement with previously reported affinity measurements . Figure 3—figure supplement 1—source data 1. Original files of western blots in . Figure 3—figure supplement 1—source data 2. Labeled western blots in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Western Blot, Purification, Concentration Assay, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Construct design of Tspan12 and Fzd4 C-terminally tagged with split GFP fragments s11 and s1–10, respectively, both downstream of a 3C protease recognition sequence. Fzd4 is additionally tagged with an N-terminal FLAG tag and Tspan12 is additionally tagged with a C-terminal 6xHis tag. The Fzd4 C-terminal PDZ ligand (ETVV) is appended after split GFP to improve expression and surface localization . ( B ) Schematic of Tspan12/Fzd4 heterodimer expression and purification. The constructs in A were co-expressed in Sf9 cells, solubilized in n -dodecyl-β-D-maltopyranoside (DDM), and co-purified on M1 anti-FLAG affinity resin followed by size exclusion chromatography. The dimer was reconstituted into nanodiscs and further purified by size exclusion followed by capture on anti-GFP nanobody resin, from which it was eluted by 3C protease. ( C ) Size exclusion traces of empty nanodiscs or Tspan12/Fzd4 dimer reconstituted into excess nanodiscs, with absorbance detected at 280 and 475 nm (GFP absorption peak), on a Superose 6 Increase column. Indicated fractions were pooled for ( D ) SDS-PAGE, imaged using StainFree imaging (above) or GFP fluorescence (below); intact GFP is SDS-resistant. Fractions on the right side of the peak (boxed) were pooled in order to exclude any potential separately-reconstituted dimers (i.e., two nanodiscs with one receptor each, linked together by the GFP moiety). ( E ) Pooled fractions were purified by GFP nanobody resin and eluted with 3C protease. The load, flowthrough, and eluate are shown. Figure 4—figure supplement 1—source data 1. Original files of gels in . Figure 4—figure supplement 1—source data 2. Labeled gels in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Construct, Sequencing, FLAG-tag, Expressing, Purification, Size-exclusion Chromatography, SDS Page, Imaging, Fluorescence, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Receptors per nanodisc in monomeric Tspan12 (mean ± SEM 1.17±0.05 Tspan12 per two MSP1D1) and heterodimeric Tspan12/Fzd4 nanodiscs (1.17±0.11 Tspan12 and 1.11±0.12 Fzd4 per two MSP1D1) as calculated from three independent samples each, with each component measured three times each by quantitative western blot. ( B ) Representative anti-Rho1D4 (top) and anti-His (bottom) western blots to quantify Tspan12-1D4 and His-MSP1D1, respectively, in monomeric Tspan12 reconstitutions. A known dilution series of purified protein was loaded in left lanes to generate a standard curve in the linear range of detection, against which dilutions of three nanodisc reconstitutions, loaded in duplicate in right lanes, were compared. ( C ) Representative anti-FLAG (top), anti-His (middle), and Neutravidin-800 (bottom) western blots to quantify FLAG-Fzd4, Tspan12-His, and biotinylated MSP1D1, respectively, in Tspan12/Fzd4 heterodimer preparations. Figure 4—figure supplement 2—source data 1. Original files of western blots in . Figure 4—figure supplement 2—source data 2. Labeled western blots in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Western Blot, Purification, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Steady-state binding curves of monomeric Tspan12∆C, monomeric Fzd4, or heterodimeric Tspan12∆C/Fzd4∆C receptors in biotinylated nanodiscs binding to dimeric or ( B ) monomeric (C93A/C95A/C131A) Norrin by biolayer interferometry (BLI). Steady-state binding signal is plotted as a percent of B max for three independent replicates (mean ± SD). Affinities and kinetic constants are reported in . ( C ) Indicated concentrations of Norrin-1D4 dimer binding to Expi293 cells transfected with Fzd4, Tspan12, or both Fzd4 and Tspan12, detected with fluorescently labeled Rho1D4 antibody and quantified by flow cytometry. Mean ± SD of three independent experiments are plotted. Co-transfection of Tspan12 increased Norrin recruitment to Fzd4-transfected cells at 0.1, 0.32, 1, and 3.2 nM Norrin (two-tailed t-test p-values of 0.00026, 0.00079, 0.0049, and 0.0018, respectively). ( D ) β-Catenin pathway activation resulting from increasing concentrations of Norrin was assessed in Fzd1/2/4/5/7/8-knockout HEK293T cells transfected with Tspan12 siRNA or increasing amounts of Tspan12 plasmid, along with Fzd4 and TopFlash luciferase reporter plasmids. Data are plotted as mean ± SD from triplicate wells are representative of three independent experiments. Figure 4—source data 1. Interference shift, cell fluorescence, and luciferase activity values used to generate .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Transfection, Labeling, Flow Cytometry, Cotransfection, Two Tailed Test, Activation Assay, Knock-Out, Plasmid Preparation, Luciferase, Fluorescence, Activity Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative biolayer interferometry (BLI) association and dissociation traces of dimeric Norrin binding to Fzd4 monomer or ( B ) Tspan12/Fzd4 heterodimer in nanodiscs. ( C ) Observed association rate constant K obs of Norrin dimer binding to Tspan12, Fzd4, or Tspan12/Fzd4 heterodimer in nanodiscs, plotted against Norrin concentration. Linear fits were used to obtain association rate constants reported in . Data represent mean ± SD for three independent replicates. ( D ) Representative BLI association and dissociation traces of monomeric Norrin (C93A/ C95A/C131A) binding to Fzd4 monomer or ( E ) Tspan12/Fzd4 heterodimer in nanodiscs. ( F ) Observed association rate constant K obs (mean ± SD) of Norrin monomer binding to Tspan12, Fzd4, or Tspan12/Fzd4 heterodimer in nanodiscs, plotted against Norrin monomer concentration. ( G ) Fzd4 surface expression on Expi293 cells transfected with empty vector, FLAG-Fzd4, or FLAG-Fzd4+Tspan12, which were then stained with M1 anti-FLAG antibody conjugated to Alexa Fluor 647. Cell fluorescence is measured by flow cytometry and plotted along with the median and interquartile range. Co-expression of Tspan12 modestly but significantly decreases surface expression of Fzd4 (Mann-Whitney test, p-value<0.0001 in each of three independent experiments).

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence, Flow Cytometry, MANN-WHITNEY

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Hypothesis: Tspan12 could enhance Norrin signaling by enhancing interactions within the Norrin-LRP5/6-Fzd4-Dvl complex, including Fzd-Dvl binding and Norrin-LRP binding. ( B ) Representative biolayer interferometry (BLI) traces of the Dvl2 DEP domain associating to and dissociating from Fzd4 in nanodiscs containing 75:20:5 POPC:Ccholesterol:PIP 2 . ( C ) Equilibrium binding of the Dvl2 DEP domain to Fzd4 monomer or Tspan12/Fzd4 heterodimer in nanodiscs; affinities ± SEM are 183±24 and 279±46 nM, respectively. ( D ) Equilibrium binding of the Dvl2 DEP domain to Fzd4 monomer or Tspan12/Fzd4 heterodimer nanodiscs, each pre-saturated with 10 nM Norrin. Binding affinities are 161±21 and 274±39 nM (mean ± SEM), respectively, determined from three independent replicates. Affinities and kinetic constants are reported in . ( E ) The LRP6 E1E2 domain fully competes with Tspan12-Norrin binding, as shown by decreased equilibrium binding of 32 nM Norrin to Tspan12 immobilized on paramagnetic particles in the presence of increasing concentrations of purified LRP6 E1E2 domain. Norrin was quantified by western blot (anti-Rho1D4; see ) and plotted as a percent of bound Norrin in the absence of LRP6 E1E2. The curve was fit to a competitive binding model using known binding affinities of 10.4 nM for Tspan12-Norrin and starting concentrations of 50 nM Tspan12 and 32 nM Norrin; the best fit reported a Norrin-LRP6 E1E2 binding affinity of 1.06 µM (95% CI 0.747–1.51 µM). Data represent mean ± SD of three replicates. ( F ) β-Catenin transcriptional activity in response to no ligand, 1 nM recombinant Norrin, or Wnt3a conditioned media (Wnt3a CM) in Fzd1/2/4/5/7/8-knockout HEK293T cells transfected with Tspan12 siRNA or indicated amount of Tspan12_pTT5 plasmid, along with Fzd4 and TopFlash luciferase reporter plasmids. Data are plotted as mean ± SD from n=3 replicate wells. Figure 5—source data 1. Interference shift, band quantification, and luciferase activity values used to generate .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay, Purification, Western Blot, Activity Assay, Recombinant, Knock-Out, Transfection, Plasmid Preparation, Luciferase

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Representative western blot of Norrin bound to Tspan12 or empty nanodiscs immobilized on paramagnetic particles in the presence of 0, 0.1, 0.4, 1.6, 6, 25, or 100 µM of purified LRP6 E1E2 domain, with both Norrin and Tspan12 detected by anti-Rho1D4 antibody. Norrin is detected within the dynamic range, as shown by concentration standards loaded to the left of the ladder. ( B ) Association and dissociation biolayer interferometry traces of purified LRP6 (residues 20–1439, including the transmembrane domain but with a truncated C-terminus, in n -dodecyl-β-D-maltopyranoside [DDM] detergent, at 32, 100, or 320 nM) binding to MBP-Norrin-loaded biosensors. ( C ) Biolayer interferometry traces of 30 nM LRP6 associating to and dissociating from MBP-Norrin-loaded biosensors, pre-equilibrated with increasing concentrations of MBP-fused Tspan12 large extracellular loop (LEL). Figure 5—figure supplement 1—source data 1. Original file of western blot in . Figure 5—figure supplement 1—source data 2. Labeled western blot in .

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Western Blot, Purification, Concentration Assay, Binding Assay, Labeling

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet: ( A ) Model: Norrin is captured by Tspan12 or heparan sulfate proteoglycans (HSPGs) and is handed off to Fzd4 for association with LRP5/6 and subsequent signaling. Norrin binding to cell-surface Fzd4 is enhanced when HSPGs concentrate Norrin at the cell surface. In contrast, Tspan12 directly and specifically delivers Norrin to co-localized Fzd4. ( B ) In the β-catenin signaling pathway, Left: Tspan12 captures Norrin and co-localizes with Fzd4, delivering Norrin to Fzd4. Middle: Likewise, RECK binds Wnt7a/b and co-localizes with Fzd via GPR124, delivering Wnt7a/b to Fzd. Right: Glypican-3 (GPC3) also binds both Fzd and Wnt to deliver Wnt to Fzd and enhance signaling. ( C ) Structurally diverse co-receptors play a similar role to Tspan12 in various pathways activated by cystine knot growth factors. Left: Neuropilin-1 (Nrp1) captures vascular endothelial growth factor A (VEGF) and co-localizes with the VEGF receptor 2 (VEGFR2) to specifically deliver VEGF to VEGFR2. Middle: The repulsive guidance molecule (RGM) binds bone morphogenic protein 2 (BMP2) as well as neogenin-1 (Neo1) to facilitate BMP signaling. Right: Betaglycan captures transforming growth factor β1 (TGF-β) and presents it to TGF-β receptor type 2 (TGFβR2).

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Binding Assay

Journal: eLife

Article Title: The co-receptor Tetraspanin12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.7554/eLife.96743

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Tspan12 (full-length, human) , This paper , RRID: Addgene_216381 , pTT5 vector; Rho-1D4 tagged; deposited at Addgene (#216381).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Binding Assay, Expressing, Control, Reporter Assay, Software