Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Afterwards cells were incubated with primary antibody against PDGFRa (goat, 1:200, AF-307-NA, R&D Systems, MN, USA), TE-7 (mouse, 1:200, Cat no CBBL271, Merck, Sigma), Pax7 (mouse, 1:50, Pax7, Developmental Studies Hybridoma Bank, IA, USA), Desmin (rabbit, 1:200, Clone D93F5, Cat no . 5332, Cell Signalling Technologies), Myosin Heavy Chain (MyHC, mouse, 1:5, MF20, DHSB), Collagen 1 (1:500, Cat no Cat. no . C2456, Sigma), alpha-smooth muscle actin (1:150, Cat no . A5228, Sigma) or pPDGFRa (Y754, rabbit, 1:20, ab5460, abcam) at 4°C overnight in blocking buffer.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Afterwards cells were incubated with primary antibody against PDGFRa (goat, 1:200, AF-307-NA, R&D Systems, MN, USA), TE-7 (mouse, 1:200, Cat no CBBL271, Merck, Sigma), Pax7 (mouse, 1:50, Pax7, Developmental Studies Hybridoma Bank, IA, USA), Desmin (rabbit, 1:200, Clone D93F5, Cat no . 5332, Cell Signalling Technologies), Myosin Heavy Chain (MyHC, mouse, 1:5, MF20, DHSB), Collagen 1 (1:500, Cat no Cat. no . C2456, Sigma), alpha-smooth muscle actin (1:150, Cat no . A5228, Sigma) or pPDGFRa (Y754, rabbit, 1:20, ab5460, abcam) at 4°C overnight in blocking buffer.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet:

Article Snippet: Incubate neurons with primary antibodies (PI3P, Z-P003, Echelon, 1:50, PI4P, Z-P004, Echelon, 1:200, or PI(4,5)P 2 , Z-A045, Echelon, 1:150 and Rab8, R5530, Sigma-Aldrich, 1:150 or TGN46, ab50595, Abcam, 1:200) in buffer A containing 5% NGS for 1 h. 41.

Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

Journal: eLife

Article Title: Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF

doi: 10.7554/eLife.67549

Figure Lengend Snippet:

Article Snippet: After blocking with PBS containing 5% normal goat/donkey serum and 0.1% Triton X-100 (blocking buffer) at RT for 1 hr, the sections were incubated at 4°C overnight with the following antibodies in the blocking buffer: mouse anti-RORC (1:800; catalog no. PP-H3925-00, Perseus Proteomics; although this antibody recognizes RORα, β and γ, RORγ is not expressed in the postnatal brain, and RORα expression generally overlaps with RORβ but is weak in the cortex [in Allen Brain Atlas]), rabbit anti-GFP (1:800; #A6455, Invitrogen), rabbit anti-Iba1 (1:500; catalog no. 019-19741, Wako), rabbit anti-5HTT (1:10,000; catalog no. 24330, ImmunoStar), goat anti-Brn2 (1:50; catalog no. sc-6029, Santa Cruz), rat anti-Ctip2 (1:200; catalog no. ab18465, Abcam), rabbit anti-Tbr1 (1:500; catalog no. ab31940, Abcam), rabbit anti-RFP (1:1000; catalog no. PM005, MBL), mouse anti-FLAG (1:1000, catalog no. F1804, Sigma-Aldrich), rabbit anti-ssDNA (1:300; catalog no. 18731, MBL), rabbit anti-RORβ (1:5000; catalog no. pAb-RORβHS-100, Diagenode), mouse anti-NeuN (1:400; catalog no. MAB377, Chemicon), rabbit anti-Cux1 (1:100, catalog no. sc-13024, Santa Cruz), rabbit anti-NRN1 (1:50; catalog no. sc-25261, Santa Cruz), and goat anti-VGF (1:50; catalog no. sc-10381, Santa Cruz).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Expressing, Sequencing, Imaging, Software, Staining

Journal: iScience

Article Title: Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution

doi: 10.1016/j.isci.2022.105802

Figure Lengend Snippet:

Article Snippet: ImmPRESS® HRP Horse Anti-Mouse IgG Polymer Detection Kit , Vector laboratories , Cat# MP-7402.

Techniques: Virus, Recombinant, Saline, Staining, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Activity Assay, Polymer, Sequencing, Expressing, Software, Imaging, Cell Culture, Microscopy, Blocking Assay

Journal: Cell reports

Article Title: VGLL4 and MENIN function as TEAD1 corepressors to block pancreatic β cell proliferation

doi: 10.1016/j.celrep.2022.111904

Figure Lengend Snippet: (A) Representative immunostaining image and quantitation of Ki67-positive β cells in wild-type mice at different ages (n = 3 mice/group). (B) Luciferase assay showing TEAD pathway activities in wild-type mouse islets at different ages (n = 3 mice/group). (C) mRNA levels of TAZ, TEAD1, and CCN2 in mouse β cells at different ages (n = 3 mice/group). (D) IPGTT showing both blood glucose and plasma insulin levels in β-Y/T KO mice and control (n = 6 mice/group). (E) IPGTT showing both blood glucose and plasma insulin levels at different time points in β-Y/T KO mice and control after 2-month high-fat diet feeding (n = 6 mice/group). (F) Immunostaining to validate TEAD1 was successfully knocked out in β-TEAD1KO (TEAD1-conditional knockout) β cells. (G) Representative immunostaining image and quantitation of Ki67-positive β cells in β-TEAD1KO and control (n = 3 mice/group). (H) Representative immunostaining image and quantitation of Ki67- positive β cells that were cultured in vitro in β-TEAD1KO and control (n = 3 mice/group). *p < 0.05; **p < 0.01; ***p < 0.001. p values are from Student’s t test. Error bars represent standard error of the mean (SEM). Scale bar, 10 μm.

Article Snippet: Primary antibodies used were TEAD1 (rabbit polyclonal, 1:200; Abcam), Ki67 (rabbit polyclonal, 1:50; Abcam), BRDU (rat polyclonal, 1:1,000; Abcam), TAZ (WWTR1) (mouse monoclonal, 1:100, Cell signaling).

Techniques: Immunostaining, Quantitation Assay, Luciferase, Knock-Out, Cell Culture, In Vitro

Journal: Cell reports

Article Title: VGLL4 and MENIN function as TEAD1 corepressors to block pancreatic β cell proliferation

doi: 10.1016/j.celrep.2022.111904

Figure Lengend Snippet: (A) Quantitation of INS1 cell number after VGLL4 overexpression on day 6. (B) Quantitation of VGLL4-overexpression INS1 cells by flow cytometry sorting analysis. (C) mRNA levels of INS1, CCN2, MAFA, GLUT2, NEUROD1, NKX6.1, PDX1, and FOXA2 after VGLL4 overexpression in INS1 cells. (D) Representative immunostaining image and quantitation of Ki67 + VGLL4 + cells in INS1 cells. (E) Representative immunostaining image and quantitation of Ki67 + VGLL4 + cells in INS2 cells (arrow). (F) Representative immunostaining image and quantitation of BRDU + VGLL4 + cells in INS2 cells (arrow head). (G) Representative immunostaining image and quantitation of Ki67-positive β cells after VGLL4 knockdown in mouse islet. (H) Representative immunostaining image and quantitation of Ki67-positive β cells after VGLL4 knockdown in human islet. *p < 0.05; **p < 0.01; ***p < 0.001. (A–G) n = at least three per condition with three technical replicates. Error bars represent standard error of the mean (SEM). p values are from Student’s t test. Scale bar, 10 μm.

Article Snippet: Primary antibodies used were TEAD1 (rabbit polyclonal, 1:200; Abcam), Ki67 (rabbit polyclonal, 1:50; Abcam), BRDU (rat polyclonal, 1:1,000; Abcam), TAZ (WWTR1) (mouse monoclonal, 1:100, Cell signaling).

Techniques: Quantitation Assay, Over Expression, Flow Cytometry, Immunostaining

Journal: Cell reports

Article Title: VGLL4 and MENIN function as TEAD1 corepressors to block pancreatic β cell proliferation

doi: 10.1016/j.celrep.2022.111904

Figure Lengend Snippet: (A) RNA-seq (TEAD1 flox/flox vs. TEAD1 −/− ) showing the changes in FOXA1, NEUROD1, NKX2.2, MAFA, NKX6.1, PDX1, Slc2a2, Mki67, FOXO1, POU3F1, CCN2, Myc, Mycn, and BMP4 in β-TEAD1KO islet. (B) mRNA levels of FZD family members in β-TEAD1KO and control islet (n = 3 mice/group). (C) mRNA level of FZD7 in mouse islets at the different ages (n = 3 mice/group). (D) ChIP-seq in mouse islet showing a TEAD1 binding peak in the promoter area of FZD7. (E) Luciferase assay using a FZD7 promoter reporter for assessing VGLL4 and MENIN repression. (F) Re-analysis of RNA-seq (MEN1 −/− vs. MEN1 flox/flox ) showing FZD7 expression in MENIN knockout islets. See also . (G) Validation of TEAD1, VGLL4, and MENIN binding to the FZD7 promoter region by ChIP experiments. (H) mRNA level of FZD7 after VGLL4 overexpression in INS1 cells. (I) Schematic representation for TEAD inhibitor (TTi) structure. N terminus of TAZ (aa12–66) was connected with FLAG tag, NLS, T2A, and mCherry. (J) mRNA level of FZD7 after MENIN overexpression or co-overexpression of MENIN and TTi in INS1 cells. (K) ChIP-seq in mouse islet showing a high TEAD1 binding peak in the promoter area of TEAD1. (L) Schematic representation for TEAD1 promoter reporter (T1R) and TEAD1 promoter reporter with MCAT mutation (T1MR). (M) The changes of luciferase activities after TEAD1 overexpression or TEAD1-VGLL4 co-expression indicated by T1R and T1mR in luciferase assay. (N) mRNA level of endogenous TEAD1 (mTEAD1) after the co-expression of human TEAD1 (hTEAD1) and VGLL4 in INS2 cells. (O) The schematic model for VGLL4/MENIN and TEAD1 interaction. *p < 0.05; **p < 0.01; ***p < 0.001. (B–E, G, H, J, M) n = at least three per condition with three technical replicates. Error bars represent standard error of the mean (SEM). p values are from Student’s t test and ANOVA.

Article Snippet: Primary antibodies used were TEAD1 (rabbit polyclonal, 1:200; Abcam), Ki67 (rabbit polyclonal, 1:50; Abcam), BRDU (rat polyclonal, 1:1,000; Abcam), TAZ (WWTR1) (mouse monoclonal, 1:100, Cell signaling).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Binding Assay, Luciferase, Expressing, Knock-Out, Over Expression, FLAG-tag, Mutagenesis

Journal: Cell reports

Article Title: VGLL4 and MENIN function as TEAD1 corepressors to block pancreatic β cell proliferation

doi: 10.1016/j.celrep.2022.111904

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were TEAD1 (rabbit polyclonal, 1:200; Abcam), Ki67 (rabbit polyclonal, 1:50; Abcam), BRDU (rat polyclonal, 1:1,000; Abcam), TAZ (WWTR1) (mouse monoclonal, 1:100, Cell signaling).

Techniques: Sequencing, Expressing, Subcloning, Recombinant, Reporter Assay, In Situ, Chromatin Immunoprecipitation, shRNA, Plasmid Preparation, Software

Journal: iScience

Article Title: Minichromosome maintenance 6 protects against renal fibrogenesis by regulating DUSP6-mediated ERK/GSK-3β/Snail1 signaling

doi: 10.1016/j.isci.2023.107940

Figure Lengend Snippet: ERK/GSK-3β/Snail1 signaling was activated in MCM6-induced renal fibrosis (A and B) Representative western blots and summarized data showing the expression of ERK, phospho-ERK, GSK-3β, phospho-GSK-3β, and Snail1 in four groups, as indicated. n = 8 mice per group. ∗∗∗p < 0.001 versus AAV-sh-Ctrl, ### p < 0.001 versus AAV-sh-Ctrl + UUO. (C and D) Representative western blots and summarized data showing the expression of ERK, phospho-ERK, GSK-3β, phospho-GSK-3β, and Snail1 in four groups, as indicated. n = 8 mice per group. ∗∗∗p < 0.001 versus AAV-OE-Ctrl, ## p < 0.01, ### p < 0.001 versus AAV-OE-Ctrl + UUO. (E and F) Representative western blots and summarized data showing the expression of ERK, phospho-ERK, GSK-3β, and phospho-GSK-3β in four groups, as indicated. TECs were transfected with si-NC or si-MCM6 for 48 h and treated with TGF-β1 for 1 h. n = 6. ∗∗∗p < 0.001 versus si-NC, ### p < 0.001 versus si-NC + TGF-β1 (1 h). (G and H) Representative western blots and summarized data showing the expression of Snail1 in four groups, as indicated. TECs were transfected with si-NC or si-MCM6 and treated with TGF-β1 for 48 h. n = 6. ∗∗∗p < 0.001 versus si-NC, ### p < 0.001 versus si-NC + TGF-β1 (48 h). (I and J) Representative western blots and summarized data showing the expression of ERK, phospho-ERK, GSK-3β, and phospho-GSK-3β in six groups, as indicated. TECs were transfected with si-NC or si-MCM6 for 48 h, pretreated with U0126 (30 μM) or SB216763 (50 μM) for 1 h, and then treated with TGF-β1 for 1 h. n = 6. ∗∗∗p < 0.001 versus si-NC, ## p < 0.01, ### p < 0.001 versus si-NC + TGF-β1 (1 h). (K) Representative western blots showing the expression of Snail1 in six groups, as indicated. TECs were transfected with si-NC or si-MCM6, pretreated with U0126 (30 μM) or SB216763 (50 μM) for 1 h, and then treated with TGF-β1 for 48 h. n = 6. All data are graphed as mean ± SEM.

Article Snippet: For some experiments, the cultured cells were pre-treated with 30 μM U0126 (Selleck Chemical, Houston, USA) or 50 μM SB216763 (Selleck Chemicals, Houston, USA) before the treatment of TGF-β1 or H/R.

Techniques: Western Blot, Expressing, Transfection

Journal: iScience

Article Title: Minichromosome maintenance 6 protects against renal fibrogenesis by regulating DUSP6-mediated ERK/GSK-3β/Snail1 signaling

doi: 10.1016/j.isci.2023.107940

Figure Lengend Snippet:

Article Snippet: For some experiments, the cultured cells were pre-treated with 30 μM U0126 (Selleck Chemical, Houston, USA) or 50 μM SB216763 (Selleck Chemicals, Houston, USA) before the treatment of TGF-β1 or H/R.

Techniques: Plasmid Preparation, Virus, Recombinant, SYBR Green Assay, Sequencing, Software

Journal: iScience

Article Title: CircMEF2C(2, 3) modulates proliferation and adipogenesis of porcine intramuscular preadipocytes by miR-383/671-3p/ MEF2C axis

doi: 10.1016/j.isci.2024.109710

Figure Lengend Snippet: Identification and expression analysis of circMEF2C(2, 3) (A) Diagram illustrating the generation of circMEF2C(2, 3) from the MEF2C gene. (B) Detection of circMEF2C(2, 3) in cDNA and gDNA using divergent and convergent primers via PCR. (C) Detection of circMEF2C(2, 3) and MEF2C expression after RNase R digestion (37°C, 50 min). (D) Confirmation of the back-splice junction (BSJ) of circMEF2C(2, 3) through Sanger sequencing. (E) Subcellular localization analysis of circMEF2C(2, 3). (F) Tissue expression profile of circMEF2C(2, 3). (G) Expression patterns of circMEF2C(2, 3) during adipogenesis. (H) Expression of circMEF2C(2, 3) in adipose tissue of MS pigs and DLY pigs. (I) Expression of circMEF2C(2, 3) in muscle tissue of MS pigs and DLY pigs.

Article Snippet: Subsequently, cDNA was synthesized from this RNase R-treated fraction using the PrimeScript RT Master kit (Takara).

Techniques: Expressing, Sequencing

Journal: iScience

Article Title: CircMEF2C(2, 3) modulates proliferation and adipogenesis of porcine intramuscular preadipocytes by miR-383/671-3p/ MEF2C axis

doi: 10.1016/j.isci.2024.109710

Figure Lengend Snippet:

Article Snippet: Subsequently, cDNA was synthesized from this RNase R-treated fraction using the PrimeScript RT Master kit (Takara).

Techniques: Recombinant, Real-time Polymerase Chain Reaction, Isolation, Negative Control, Software

Journal: Cell Reports Medicine

Article Title: Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus

doi: 10.1016/j.xcrm.2024.101647

Figure Lengend Snippet: FLVCR1, responsible for CH in humans, is highly expressed by NPCs during development (A) Sonographic examinations of 32 + 4 weeks of pregnancy showing extreme microcephaly with anechoic skull and no evidence of cerebral tissue in a human fetus carrying the c.160delC, p.Arg54GlyfsTer59 mutation in the FLVCR1 gene. (B) Family tree of the affected fetus. (C) FLVCR1a expression data from developing human brain extracted from the dataset GSE25219 of the Human BrainSpan Atlas. This dataset consists of RNA sequencing and exon microarrays obtained at sequential developmental stages of the human brain. Heatmap indicates low (blue) and high (red) expression values. Gray color: no available data. PCW, post-conception weeks; A1C, auditory cortex; AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; IPC, posterior inferior parietal cortex; ITC, inferior temporal cortex; M1C, primary motor cortex; MD, mediodorsal nucleus of the thalamus; MFC, medial pre-frontal cortex; OFC, orbital prefrontal cortex; S1C, primary somatosensory cortex; STC, superior temporal cortex; STR, striatum; V1C, primary visual cortex; VFC, ventrolateral prefrontal cortex. (D) FLVCR1a staining (red) in 11 post-conception weeks (PCW) human brain sections shows expression in RGCs (PAX6+ and SOX2+ cells) and IPs (TBR2+ cells). Scale bar: 500 μm. Magnified images of the insets on the left are shown on the right. Scale bar: 100 μm. (E) Western blot analysis of FLVCR1a expression in the brains isolated from E12.5 to E18.5 embryos, 2 days (P2) and 3-month-old Flvcr1-myc mice. An anti-Myc-Tag antibody was used to detect endogenous FLVCR1a. A representative image is shown. (F) Flvcr1a mRNA levels in “FlashTag”-labeled cell populations in the developing mouse cortex. The different isolation time points correspond to specific populations: 1 h = RGCs; 10 h = IPs; 24 h = newborn neurons; 4 days = neurons. Data represent mean ± SEM. See also Figure S1 and .

Article Snippet: Cells were subsequently incubated with primary antibodies: FLVCR1 primary antibody (Santa Cruz Biotechnology, Dallas, TX USA, catalog n° sc-390100; 1:50), rabbit FLVCR1a (Proteintech, catalog n° 26841), VDAC (Abcam, catalog n° ab154856), IP3R3 (BD biosciences, catalog n° 610312), GRP75 (Proteintech, catalog n°14887), Sigma 1R (Sigma Aldrich, catalog n° HPA018002).

Techniques: Mutagenesis, Expressing, RNA Sequencing, Staining, Western Blot, Isolation, Labeling

Journal: Cell Reports Medicine

Article Title: Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus

doi: 10.1016/j.xcrm.2024.101647

Figure Lengend Snippet: FLVCR1a interacts with the IP3R3-VDAC complex (A) STRING analysis of the top 50 FLVCR1a interactors. (B) Gene Ontology term analysis for subcellular compartments of the top 50 FLVCR1a interactors. (C) Subcellular fractioning of HeLa cells. H, homogenate; ER, endoplasmic reticulum; MAMs, mitochondria-associated membranes; Mito, pure mitochondria. (D) PLA was performed in HeLa cells using the FLVCR1a and VDAC1 antibodies (FLVCR1a-ATP5i pair was used as a negative control), or the FLVCR1a and IP3R3 antibodies (FLVCR1a-PDI pair was used as a negative control), or the FLVCR1a and GRP75 antibodies (FLVCR1a-Laminin pair was used as a negative control). (non parametric Mann-Whitney U test; ∗∗∗∗ = p < 0.0001.) (E) Immunoprecipitation assay to detect the interaction between IP3R3-GFP and FLVCR1a-Myc. The protein complex was immune-precipitated using anti-GFP antibody, and the eluted proteins were detected by immunoblotting using either an anti Myc-Tag or an anti-GFP antibody. The vector expressing GFP alone was used as a negative control. (F) Immunoprecipitation assay to detect the interaction between FLVCR1a-Myc and IP3R3-GFP. The protein complex was immune-precipitated using anti-Myc-Tag antibody, and the eluted proteins were detected by immunoblotting using either an anti-GFP or an anti Myc-Tag antibody. (G) Immunoprecipitation assay to detect the interaction between FLVCR1a-Myc and VDAC-HA. The protein complex was immune-precipitated by an anti-Myc-Tag antibody, and the eluted proteins were detected by immunoblotting using either an anti-HA or an anti Myc-Tag antibody. (H) Immunoprecipitation of endogenous FLVCR1a from HEK293 cells followed by immunoblotting of the eluted proteins by an anti-VDAC antibody. (I) Immunoprecipitation of endogenous VDAC from E18.5 brains collected from the Flvcr1-myc embryos followed by immunoblotting using an anti-myc-Tag antibody. See also Figures S7 and .

Article Snippet: Cells were subsequently incubated with primary antibodies: FLVCR1 primary antibody (Santa Cruz Biotechnology, Dallas, TX USA, catalog n° sc-390100; 1:50), rabbit FLVCR1a (Proteintech, catalog n° 26841), VDAC (Abcam, catalog n° ab154856), IP3R3 (BD biosciences, catalog n° 610312), GRP75 (Proteintech, catalog n°14887), Sigma 1R (Sigma Aldrich, catalog n° HPA018002).

Techniques: Negative Control, MANN-WHITNEY, Immunoprecipitation, Western Blot, Plasmid Preparation, Expressing

Journal: Cell Reports Medicine

Article Title: Dysregulation of FLVCR1a-dependent mitochondrial calcium handling in neural progenitors causes congenital hydrocephalus

doi: 10.1016/j.xcrm.2024.101647

Figure Lengend Snippet:

Article Snippet: Cells were subsequently incubated with primary antibodies: FLVCR1 primary antibody (Santa Cruz Biotechnology, Dallas, TX USA, catalog n° sc-390100; 1:50), rabbit FLVCR1a (Proteintech, catalog n° 26841), VDAC (Abcam, catalog n° ab154856), IP3R3 (BD biosciences, catalog n° 610312), GRP75 (Proteintech, catalog n°14887), Sigma 1R (Sigma Aldrich, catalog n° HPA018002).

Techniques: Control, Purification, Marker, Recombinant, Sterility, Protease Inhibitor, Bicinchoninic Acid Protein Assay, ATP Bioluminescent Assay, Reverse Transcription, Transfection, Imaging, SYBR Green Assay, Plasmid Preparation, shRNA, Software, Variant Assay

Journal: Cancer cell

Article Title: Mitochondrial reprogramming underlies resistance to BCL-2 inhibition in lymphoid Malignancies

doi: 10.1016/j.ccell.2019.08.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunohistochemistry and image acquisition Immunohistochemical staining (IHC) for MCL1 (Clone 22, 1:100, Santa Cruz), Acetyl-CoA Carboxylase (Clone C83B10, 1:50, Cell Signaling), Phospho-Acetyl-CoA Carboxylase (Clone D7D11, 1:50, Cell Signaling) or AMPKα (clone D5A2, 1:50, Cell Signaling) was performed using an automated staining system (BondRX, Leica Biosystems, Buffalo Grove, IL) according to the manufacturer’s protocol and as previously described ( Roemer et al., 2016 ).

Techniques: Recombinant, Cell Viability Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Picogreen Assay, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, CRISPR, Knock-Out, Software

Journal: Cell reports

Article Title: Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome

doi: 10.1016/j.celrep.2020.108179

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-nucleolin , Abcam , Cat#ab22758; RRID: AB_776878.

Techniques: Recombinant, Transfection, Sequencing, Transformation Assay, Software

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: SLAMF1 is enriched in the Rab11-positive ERCs in unstimulated macrophages, and SLAMF1 expression is induced by LPS and several other TLR ligands in primary human monocytes and macrophages. (A) Monocytes, macrophages, and differentiated THP-1 cells stained with antibodies against SLAMF1 (green) and GM130 (red) and imaged by confocal microscopy. (B) 3D model of cis-Golgi (GM130) and SLAMF1 in THP-1 cells. Z stacks from the GM130 and SLAMF1 channels were obtained using high-resolution confocal microscopy followed by 3D modeling in IMARIS software. (C) Macrophages stained for SLAMF1 and Rab11 (ERC marker). Representative image. Overlapping pixels for SLAMF1 and Rab11 are shown in the white overlap. tM1 = 0.683 ± 0.08 (mean with SD) for z stacks of ERCs as ROIs (30 ROIs analyzed per donor) where tM1 was the Manders’s colocalization coefficient with thresholds calculated in the Coloc 2 Fiji plugin with anti-SLAMF1 staining as first channel. (D) Macrophages costained for SLAMF1 and EEA1. (E) Macrophages costained for SLAMF1 and LAMP1. Colocalization accessed for z stacks for at least 30 cells for each experiment (four total) showing no colocalization for markers in both D and E. (F) Flow cytometry analysis of SLAMF1 surface expression by primary macrophages and differentiated THP-1 cells. Cells were costained for SLAMF1 and CD14 and gated for CD14-positive cells (primary cells) or stained for SLAMF1 (THP-1 cells). (G) Flow cytometry analysis of SLAMF1 surface expression by human macrophages stimulated by ultrapure K12 LPS (100 ng/ml) for 2, 4, and 6 h. (H) Western blot analysis of lysates from primary human macrophages stimulated by LPS for 2, 4, and 6 h. Graphs present mean values for three biological replicates with SD. Molecular weight is given in kilodaltons. (I and J ) Quantification of SLAMF1 mRNA expression by qPCR in monocytes (I) and macrophages (J) stimulated by TLRs’ ligands FSL-1 (20 ng/ml), K12 LPS (100 ng/ml), and CL075 (1 μg/ml; both I and J) as well as R848 (1 μg/ml), Pam3Cys (P3C; 1 μg/ml), or K12 E. coli particles (20/cell; I only). Results are presented as means with SD. Statistical significance between groups was evaluated by a two-tailed t test. *, P < 0.01. Results are representative of at least four independent experiments/donors (A–H) or combined data for at least three donors (I and J).

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Expressing, Staining, Confocal Microscopy, Software, Marker, Flow Cytometry, Western Blot, Molecular Weight, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: Knockdown of SLAMF1 in macrophages results in strongly reduced TLR4-mediated IFNβ mRNA expression and protein secretion as well as some decrease of TNF, IL-6, and CXCL10 secretion. (A and B) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in THP-1 cells (A) and macrophages (B) treated by 100 ng/ml ultrapure K12 LPS. (C and D) IFNβ and TNF secretion levels by THP-1 cells (C) and macrophages (D) in response to LPS (4 and 6 h) assessed by ELISA. (E and F) Secretion levels of IL-1β, IL-6, IL-8, and CXCL-10 (6 h LPS) analyzed by multiplex assays. Data are presented as means with SD for combined data from three independent experiments (A, C, and E), for three biological replicates from one of six donors (B and D), or one of three donors (F). *, P < 0.01.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Multiplex Assay

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: SLAMF1 silencing in macrophages impairs TLR4-mediated phosphorylation of TBK1, IRF3, and TAK1. Western blotting of lysate macrophages treated with a control nonsilencing oligonucleotide or SLAMF1 -specific siRNA oligonucleotides and stimulated with 100 ng/ml LPS. The antibodies used are indicated in the figure. An antibody toward SLAMF1 was used to control for SLAMF1 silencing, and GAPDH was used as an equal loading control. Same GAPDH controls are presented for pTBK1, total TBK1, and phospho-p38MAPK, for total IRF3 and total TAK1, and for pTAK1 and pIκBα because they were probed on the same membranes. Western blots are representative of one of five donors. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Western Blot, Molecular Weight, Software

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: Lentiviral transduction of SLAMF1 in macrophages results in the increase of IRF3 and TBK1 phosphorylation in response to LPS and upregulation of IFNβ and TNF expression. (A) Quantification of SLAMF1 , IFNβ , and TNF mRNA expression by qPCR in macrophages transduced by Flag-tagged SLAMF1 coding or control virus and treated by LPS. The qPCR data are presented as means and SD for three biological replicates of one of three experiments. Significance was calculated by two-tailed t tests. *, P < 0.01. (B) Western blots showing LPS-induced phosphorylation of signaling molecules in cells transduced with the SLAMF1-expressing virus versus control virus. Dividing lines were added where the time point of 4 h was excised. The same GAPDH controls are presented for total IRF3 and total IκBα and for pIκBα and pTAK1 because they were probed on the same membranes. Molecular weight is given in kilodaltons. Graphs (right) show quantifications of protein levels relative to GAPDH levels obtained with Odyssey software.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Transduction, Expressing, Two Tailed Test, Western Blot, Molecular Weight, Software

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: SLAMF1 regulates TRAM recruitment to E. coli phagosomes. (A) SLAMF1 costaining with TRAM, EEA1, or LAMP1 in primary macrophages coincubated with E. coli pHrodo particles for indicated time points. SLAMF1 (green), E. coli (blue), and TRAM, EEA1, or LAMP1 (red) are shown. The data shown are representative of one out of four donors. Bars, 10 μm. (B and C) TRAM and SLAMF1 MIs on E. coli phagosomes upon SLAMF1 silencing (B) or simultaneous Rab11a and Rab11b silencing (C) in primary human macrophages quantified from xyz images. The scatter plots are presented as median values of TRAM voxel intensity, and numbers of phagosomes are shown at the top. The nonparametric Mann-Whitney test was used to evaluate statistical significance. *, P < 0.01; ***, P ≤ 0.0001. Human macrophages were incubated with E. coli particles for indicated time points, fixed, and costained for SLAMF1 and TRAM, normal rabbit (rIgG), or mouse IgG (mIgG). The data shown are representative for one out of five (B) or four (C) donors.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: MANN-WHITNEY, Incubation

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: SLAMF1 interacts with TRAM protein. (A) Endogenous IPs using specific anti-SLAMF1 mAbs from macrophages stimulated by LPS. (B) Endogenous IPs using anti-TRAM polyclonal antibodies from macrophages stimulated by LPS. (C) TRAM Flag -precipitated SLAMF1 and SLAMF1ct was needed for interaction with TRAM. (D) Coprecipitation of TRAM deletion mutants: TIR domain (68–235), short TRAM TIR domain (68–176 aa), and N-terminal (1–68 aa) or C-terminal (158–235 aa) domains with SLAMF1 protein. (E) Coprecipitation of TRAM deletion mutants containing the N-terminal part of TRAM TIR domain with SLAMF1. (F) Coprecipitation of SLAMF1 Flag deletion mutants with TRAM YFP . (G) Coprecipitation of human SLAMF1 Flag with human TRAM YFP and of mouse SLAMF1 Flag with mouse TRAM EGFP . Black dashed lines indicate that intervening lanes have been spliced out. (H) Human SLAMF1 cytoplasmic tail coprecipitation with TRAM YFP with or without amino acid substitutions at 321–324. Graphs under C–F summarize the IPs’ results. Indicated constructs were transfected to HEK293T cells, and anti-Flag agarose was used for the IPs. For endogenous IPs, specific SLAMF1 or TRAM antibodies were covalently coupled to beads. At least three independent experiments were carried out for anti-Flag IPs, and five independent experiments were carried out for the endogenous IPs, and one representative experiment is shown for each. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Construct, Transfection, Molecular Weight, Western Blot

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: SLAMF1 interacts with all class I Rab11 FIPs. (A) Anti-Flag IPs for Rab11a Flag with EGFP-tagged Rab11FIPs (1–5) and SLAMF1. (B) Schematic figure for class I and class II Rab11 FIPs domain structure. C2, phospholipid-binding C2 domain; EF, EF-hand domain; PRR, proline-rich region; RBD, Rab11 binding domain. (C) Homologous protein sequence in class I FIPs, which follow the C2 domain. Identical amino acids in all three class I FIPs are highlighted. (D) Coprecipitation of SLAMF1 Flag with FIP2 EGFP WT or FIP2 deletion mutant lacking the C2 domain (ΔC2). (E and F) Coprecipitation of untagged SLAMF1 with FIP2 Flag (1–512 aa) and Flag-tagged FIP2 deletion mutants in anti-Flag IPs in the absence (E) or presence (F) of overexpressed Rab11 CFP . (G) Quantification of coprecipitations in E and F between SLAMF1 and FIP2 Flag variants correlated with the amount of Flag-tagged protein on the blot and Flag-tagged protein sizes. Error bars represent means ± SD for three independent experiments. (H) Coprecipitation of FIP2 Flag with SLAMF1 and Rab11a WT, Rab11a Q70L mutant (QL), or Rab11a S25N mutant (SN). (I) Coprecipitation of SLAMF1 Flag deletion mutants with FIP2 EGFP . Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate. (J) Scheme for FIP2- and TRAM-interacting domains in SLAMF1ct. The results are representative of at least three independent experiments.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Binding Assay, Sequencing, Mutagenesis, Molecular Weight, Western Blot

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: TRAM acts as a bridge between the SLAMF1 and TLR4 signaling complex. (A and B) Coprecipitations of SLAMF1 Flag with TLR4 Cherry (A) or TRIF HA (B) with or without TRAM YFP overexpression. (C) Coprecipitation of TLR4 Flag with SLAMF1 with or without TRAM YFP overexpression. (D) TLR4 Flag interaction with TRAM YFP and TRIF HA with or without SLAMF1 coexpression. (E) Coprecipitation of SLAMF1 with or without TRIF HA in the presence of TRAM YFP by TLR4 Flag . Indicated constructs were transfected to HEK293T cells. pDuo-CD14/MD-2 vector was cotransfected to all wells (A and C–E). Anti-Flag agarose was used for IPs. At least three independent experiments were performed. Molecular weight is given in kilodaltons. WB, Western blot; WCL, whole-cell lysate.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Over Expression, Construct, Transfection, Plasmid Preparation, Molecular Weight, Western Blot

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: TRAM and SLAMF1 are essential for the killing of E. coli by human macrophages. (A) Flow cytometry analysis of dihydrorhodamine 123 (DHR-123) fluorescence to access ROS activation in control siRNA or SLAMF1 siRNA human macrophages upon stimulation by E. coli red pHrodo particles. One of three experiments shown. (B and C) Bacterial killing assays by SLAMF1-silenced and control THP-1 cells (B) as well as TRAM KO and control THP-1 cells (C) infected with a DH5α strain at MOI 40. (D and E) Western blot analysis of pAkt (S473) and pIRF3 (S396) levels induced by E. coli particles in THP-1 WT and TRAM KO cells (D) as well as SLAMF1-silenced or control oligonucleotide–treated cells (E). Graphs (right) on Western blotting show quantification of protein levels relative to β-tubulin obtained with Odyssey software. (F) Western blot showing phospho-(S396) IRF3 and phospho-(S473) Akt levels in lysates of THP-1 cells coincubated with E. coli particles for 1 h in the presence or absence of TBK1-IKKε inhibitor (MRT67307), pan-Akt allosteric inhibitor (MK2206), or DMSO. Molecular weight is given in kilodaltons. (G) Bacterial killing assays by THP-1 cells with DMSO (<0.01%), 1 μM Akt inhibitor MK2206, or 2 μM TBK1-IKKε inhibitor MRT67307 upon infection by DH5α at MOI 40. Percent killing was calculated as 100 − (number of CFUs at time X/number of CFUs at time 0) × 100 for average values of technical replicates, and each dot on the graphs in B, C, and G represents a biological replicate from three independent experiments. Median values are shown by lines. Statistical significance was calculated by a Mann-Whitney nonparametric test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Flow Cytometry, Fluorescence, Activation Assay, Infection, Western Blot, Software, Molecular Weight, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: SLAMF1 is required for TLR4-mediated TRAM-TRIF–dependent signaling in human macrophages

doi: 10.1083/jcb.201707027

Figure Lengend Snippet: Primers used for cloning of full-size or deletion mutants of SLAMF1, TRAM, TLR4, and Rab11 FIP2.

Article Snippet: The following primary antibodies were used: rabbit anti–TICAM-2/TRAM (GTX112785) from Genetex; rabbit mAb anti–human SLAMF1/SLAMF1 (10837-R008-50) from Sino Biological Inc.; mouse anti-GAPDH (ab9484) and rabbit anti–phospho-IRF3 Ser386 (ab76493) from Abcam; rabbit anti–phospho-Akt Ser473 (D9E; 4060), phospho-IRF3 Ser396 (4D4G; 4947), IkB-α (44D4; 4812), phospho–IkB-α (14D4; 2859), p38MAPK (9212), phospho-p38MAPK (Thr180/Tyr182; D3F9; 4511), TBK1/NAK (D1B4; 3504), phospho-TBK1/NAK (Ser172; D52C2; 5483), phospho-TAK1 (T184/187; 90C7; 4508), TAK1 5206, phospho–stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185; 81E11; 4668), anti-DYKDDDDK tag (D6W5B)/Flag tag (14793), anti-MyD88 (D80F5; 4283), and phospho-STAT1 (Tyr701; D4A7; 7649) from Cell Signaling Technology; rabbit anti–total IRF3 (FL-425; sc-9082) and proliferating cell nuclear antigen (PCNA; FL-261; sc-7907) were from Santa Cruz Biotechnology, Inc.; Living Colors rabbit anti–full-length GFP polyclonal antibodies (632592) from Takara Bio Inc.; 4G10 platinum antiphosphotyrosine antibody biotin conjugated (16-452) from EMD Millipore; and mouse anti-GST antibodies (SAB4200237) and monoclonal mouse ANTI-FLAG M2 antibodies (F1804-200UG) from Sigma-Aldrich.

Techniques: Clone Assay, Sequencing, Mutagenesis, Plasmid Preparation, Construct

Journal: Current biology : CB

Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

doi: 10.1016/j.cub.2019.04.072

Figure Lengend Snippet: (A) RT-PCR for trpm8 (236 bp) from cDNA extracted from stage 24 whole embryo or dissected spinal cord. +/− RT, in the presence or absence of the reverse transcriptase, respectively, during conversion of isolated mRNA into cDNA. (B) In situ hybridization for trpm8 in stage 24 embryos showing specific labeling in brain and spinal cord. (C) Western blot assays from egg, stage 24 wild-type whole embryo or from stage 40 whole larva, control morpholino (MO) or TRPM8-translation-blockingMO 1 (TRPM8-tbMO1) lysates. Predicted TRPM8 molecular weight [MW]: 132 kDa. Shown are representative examples of one of 3 independent experiments. β-tubulin was used as loading control. (D) Immunostained transverse section of stage 25 spinal cord (outlined). D, dorsal; V, ventral; scale bar, 20 μm; arrows indicate TRPM8 clusters in ventral neuron domains. NCAM labeling was used as counterstaining. (E) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged at 1 Hz for 90 s. Either 100 μM (−)-menthol or vehicle (0.05% DMSO)was added after 35 s of imaging and recording continued for another 60 s. Images show a menthol-responsive ventral neuron before (left, control) and after (right) addition of (−)-menthol. Colored scale shows fluorescence intensity in arbitrary units. Traces show the changes in fluorescence for the indicated cell (arrow) in both trials. (F) Stage 24 ventral spinal cord from wild-type embryos was Ca 2+ imaged in 30-min intervals at cold (14.5°C) and warm (26.5°C) temperatures in the absence (vehicle, 0.1% DMSO) or presence of 10 μM AMTB, TRPM8 inhibitor. Scatterplots show changes in Ca 2+ spike frequency when switching temperatures in individual spinal neurons and geometric mean (black lines) from N = 3 ventral spinal cords per condition (n of neurons analyzed: DMSO, 62; AMTB, 71). Teal circles represent neurons with higher spike frequency at 14.5°C, magenta circles represent neurons with higher spike frequency at 26.5°C, and black circles represent neurons with no change in spike frequency across temperatures; ****p < 0.0001, comparison within treatments Wilcoxon matched-pairs signed rank, two-tailed test. (G) RT-PCR from cDNA collected from stage 46 larvae previously injected with 2.5 pmol standard control morpholino (Control-MO) or TRPM8-splicing-blocking morpholino (TRPM8-sbMO) shows that trpm8 mature transcript (349 bp) is not detected in TRPM8-sbMO animals. odc : ornithine decarboxylase (101 bp) as positive control. (H) TRPM8-sbMO or Control-MO containing spinal cord from stage 24 embryos were Ca 2+ -imaged for 30 min at cold temperature (14.5°C). Graph shows individual (scatterplots) and geometric mean (black lines) Ca 2+ spike frequency from N = 3 ventral spinal cords pergroup (n of neurons analyzed: Control-MO, 81; TRPM8-sbMO, 64), ****p < 0.001, Kolmogorov-Smirnov, two-tailed test. See also and .

Article Snippet: For protein loading control, PVDF membranes were stripped in stripping buffer (0.2 M glycine-HCl buffer, pH 2.5, 0.05% Tween) for 20 min then re-probed overnight with anti-β-tubulin mouse monoclonal antibody (1:50 in 5% milk, Developmental Studies Hybridoma Bank), followed by HRP-conjugated secondary antibody (Jackson ImmunoResearch; 1:10,000) and visualized as described above.

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Isolation, In Situ Hybridization, Labeling, Western Blot, Control, Molecular Weight, Imaging, Fluorescence, Comparison, Two Tailed Test, Injection, Blocking Assay, Positive Control

Journal: Current biology : CB

Article Title: Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function

doi: 10.1016/j.cub.2019.04.072

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For protein loading control, PVDF membranes were stripped in stripping buffer (0.2 M glycine-HCl buffer, pH 2.5, 0.05% Tween) for 20 min then re-probed overnight with anti-β-tubulin mouse monoclonal antibody (1:50 in 5% milk, Developmental Studies Hybridoma Bank), followed by HRP-conjugated secondary antibody (Jackson ImmunoResearch; 1:10,000) and visualized as described above.

Techniques: Recombinant, Western Blot, In Situ, Luciferase, Reporter Assay, Reverse Transcription, cDNA Synthesis, Isolation, Sequencing, Control, Knockdown, Software