Journal: International Journal of Molecular Sciences

Article Title: Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats

doi: 10.3390/ijms24021206

Figure Lengend Snippet: E2 increased Klotho expression in the hippocampus of ovariectomized (OVX) rats and cultured hippocampal neurons. Experimental design ( A ). Representative images of KL immunostaining (red) in the hippocampal CA1 area of sham, OVX, and E2-treated OVX (OVX + E2) rats (48 h after vehicle or E2 injection ( B , C ); daily vehicle or E2 injection for 7 days ( F – H )). Quantification of fluorescence intensity of KL staining in the hippocampal CA1 area (48 h ( D ); 7 d ( I )). Western blot showing the relative KL protein levels in the hippocampus of sham, OVX, and OVX + E2 rats (48 h ( E ); 7 d ( J ); n = 6). The cultures were treated with the vehicle (Con), 10 nM E2, and E2 + 1 μM ICI (ICI182,780, estrogen receptor inhibitor) at Div13 for 48 h. Western blot result of hippocampal lysate prepared from primary hippocampal culture ( K ). Immunostaining of cultured hippocampal neurons with antibodies specific to KL and MAP2 in Con, E2, and E2 + ICI neurons ( L ). KL staining (green, ( L1 , L4 , L7 )), MAP2 staining (blue, ( L2 , L5 , L8 )), and merge of KL and MAP2 ( L3 , L6 , L9 ). Quantification of KL-positive clusters in cultured hippocampal neurons ( N ). Immunostaining of cultured hippocampal neurons with antibodies specific to Vglut1 and MAP2 in Con, E2, and E2 + ICI neurons ( M ). Vglut1 staining (red, ( M1 , M4 , M7 )), MAP2 staining (blue, ( M2 , M5 , M8 )), and merge of Vglut1 and MAP2 ( M3 , M6 , M9 ). The number of Vglut1-positive clusters in cultured hippocampal neurons ( O ). SO, stratum oriens of CA1 area; SP, stratum pyramidale of CA1 area; SR, stratum radiatum of CA1 area; Con, control. Data are shown as the mean ± SEM. D with the t -test, and others with one-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents were used: β-estradiol 3-benzoate (E8515, Sigma-Aldrich, St. Louis, MO, USA), sesame oil (S3547, Sigma-Aldrich, St. Louis, MO, USA), β-estradiol suitable for cell culture (E2257, Sigma-Aldrich, St. Louis, MO, USA), ICI182,780 (estrogen receptor antagonist, 1047, TOCRIS, Bristol, UK), letrozole (aromatase inhibitor, 4382, TOCRIS, Bristol, UK), and primary antibodies (Klotho (AF1819, R&D Systems, Minneapolis, MN, USA, immunostaining and Western blot), Klotho (ab203576, Abcam, Cambridge, UK Western blot), vesicular glutamate transporter (Vglut1, AB5905, Millipore, Bredford, MA, USA), and MAP2 (sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA)).

Techniques: Expressing, Cell Culture, Immunostaining, Injection, Fluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats

doi: 10.3390/ijms24021206

Figure Lengend Snippet: Klotho played an essential role in the E2-mediated increase in the number of Vglut1-positive clusters in hippocampal neurons. Primary cultured hippocampal neurons were transfected with a vector encoding scrambled control shRNA (sh-Scr, ( A1 – A4 )) or Klotho shRNA (sh-KL, ( A5 – A8 )) at Div10, and the neurons were fixed for double-immunostaining with anti-KL (red) and anti-MAP2 (blue) antibodies at Div14. The dashed lines in ( A6 ) show the dendrites of the non-transfected neuron, which did not express Klotho shRNA-GFP ( A ). Quantification of KL expression in cultured hippocampal neurons ( B ). Two different Klotho shRNAs, sh-KL #1 and sh-KL# 2, were designed. Vectors encoding sh-Scr and sh-KL were introduced into the hippocampal neurons by electroporation at the time of plating, and the Western blot result ( C ) showed that the expression of shRNA #1 and #2 reduces klotho (130 kDa) expression effectively. We used sh-KL #1 in subsequent experiments. Cultured hippocampal neurons were transfected with a vector encoding sh-Scr ( D1 – D8 ) and sh-KL ( D9 – D16 ) at Div10, and the cultures at Div13 were treated with the vehicle ( D1 – D4 , D9 – D12 ) or 10 nM E2 ( D5 – D8 , D13 – D16 ) for 48 h before fixing for double-immunostaining with antibodies specific to Vglut1 (red) and MAP2 (blue; D ). Quantification of Vglut1-positive clusters ( E ). One-way and two-way ANOVA followed by Tukey’s test. Data are shown as the mean ± SEM. ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents were used: β-estradiol 3-benzoate (E8515, Sigma-Aldrich, St. Louis, MO, USA), sesame oil (S3547, Sigma-Aldrich, St. Louis, MO, USA), β-estradiol suitable for cell culture (E2257, Sigma-Aldrich, St. Louis, MO, USA), ICI182,780 (estrogen receptor antagonist, 1047, TOCRIS, Bristol, UK), letrozole (aromatase inhibitor, 4382, TOCRIS, Bristol, UK), and primary antibodies (Klotho (AF1819, R&D Systems, Minneapolis, MN, USA, immunostaining and Western blot), Klotho (ab203576, Abcam, Cambridge, UK Western blot), vesicular glutamate transporter (Vglut1, AB5905, Millipore, Bredford, MA, USA), and MAP2 (sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA)).

Techniques: Cell Culture, Transfection, Plasmid Preparation, shRNA, Double Immunostaining, Expressing, Electroporation, Western Blot

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a HEK293T cells co-expressing Myc-Plk4 and GFP-Cep131 were immunoprecipitated using Myc-magnetic beads, and the protein levels were observed by immunoblotting. b Endogenous Plk4 extracted from U2OS cells was immunoprecipitated by anti-Plk4, and total and pulled down proteins were observed by immunoblotting. c Bacterially generated GST-Plk4-WT or KD were incubated with His-Cep131 (1-911 aa) and pulled down using GSH-agarose. d , e HEK293T cells co-expressing Myc-Plk4 and HA-Cep131 deletions, including #mut2, #mut2+3, and #mut3, as illustrated in e , were immunoprecipitated, and protein levels were observed. f Alignment of amino acids from 513–542 in human Cep131 corresponded to several other species. Plk4-binding motif (PBM) is highlighted in red. Asterisk, fully conserved residue; colon, conserved residue; period, semi-conserved residue. A Coomassie (CBB) staining use as a loading control in b – d

Article Snippet: Rabbit polyclonal antibodies against Cep131 (A301-415A, immunofluorescence (IF) 1:100, immunoblotting (IB) 1:2,000; Bethyl Laboratories, Montgomery, TX, USA), STIL (A302-441A, IF 1:100, IB 1:1,000; Bethyl Laboratories) (ab89314; immunoprecipitation (IP) 2 mg/ml; Abcam, Cambridge, UK;), Plk4 (ab1373398, IB 1:1,000, IP 2 mg/ml; Abcam), γ-tubulin (T5192, IF 1:500, IB 1:3,000; Sigma-Aldrich, St. Louis, MO, USA) HA-tag (Y-11, sc-805, IF 1:100, IB 1:2,000; Santa Cruz Biotechnology; Dallas, TX, USA), and GFP-tag (FL, sc-8334, IB 1:2,000; Santa Cruz Biotechnology), as well as mouse monoclonal antibodies against centrin (20H5, 04-1624, IF 1:100; EMD Millipore, Billerica, MA, USA), SAS-6 (91.390.21, sc-81431, IF 1:100; Santa Cruz Biotechnology), ninein (clone 79-160, MABT29, IF 1:200; EMD Millipore), γ-tubulin (T6557, IF 1:500, IB 1:3,000; Sigma-Aldrich), and c-Myc (9E10, sc-40, IB 1:2,000; Santa Cruz Biotechnology) were used and purchased as indicated.

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, Western Blot, Generated, Incubation, Binding Assay, Residue, Staining, Control

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a Alignment of amino acids in human Cep131 and several other species. The phosphorylation sites at residues S21 by Plk4 are highlighted in red. Asterisk, fully conserved residue; colon, conserved residue; period, semi-conserved residue. b Peptide competition assay to verify the specificity of anti-Cep131-pS21 antibody. Lysates from HEK293T cells expressing Myc-Plk4 with HA-Cep131-WT or S21A were incubated with non- or phosphopeptides targeting Cep131-S21. After incubation, phosphorylation status was analyzed by immunoblotting using Cep131-pS21 antibody. c HEK293T cells co-expressing HA-Cep131-WT with Myc-Plk4-WT or KD were immunoprecipitated using HA-magnetic beads, followed by incubation with λ-phosphatase at 30 °C for 30 min. Phosphorylation status was then observed by immunoblotting using anti-Cep131-pS21 antibody. d Immunofluorescence analysis of co-localization of phosphorylated Cep131 at the centriole. U2OS cells were treated with shGL2 (control) or shCep131 and were co-stained for Cep131-pS21 (red) and Cent (green). e Quantification of Cep131-pS21-positive cells. Error bars represent means ± SEM from three independent experiments ( N > 300 for each experiment). ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in b , c

Article Snippet: Rabbit polyclonal antibodies against Cep131 (A301-415A, immunofluorescence (IF) 1:100, immunoblotting (IB) 1:2,000; Bethyl Laboratories, Montgomery, TX, USA), STIL (A302-441A, IF 1:100, IB 1:1,000; Bethyl Laboratories) (ab89314; immunoprecipitation (IP) 2 mg/ml; Abcam, Cambridge, UK;), Plk4 (ab1373398, IB 1:1,000, IP 2 mg/ml; Abcam), γ-tubulin (T5192, IF 1:500, IB 1:3,000; Sigma-Aldrich, St. Louis, MO, USA) HA-tag (Y-11, sc-805, IF 1:100, IB 1:2,000; Santa Cruz Biotechnology; Dallas, TX, USA), and GFP-tag (FL, sc-8334, IB 1:2,000; Santa Cruz Biotechnology), as well as mouse monoclonal antibodies against centrin (20H5, 04-1624, IF 1:100; EMD Millipore, Billerica, MA, USA), SAS-6 (91.390.21, sc-81431, IF 1:100; Santa Cruz Biotechnology), ninein (clone 79-160, MABT29, IF 1:200; EMD Millipore), γ-tubulin (T6557, IF 1:500, IB 1:3,000; Sigma-Aldrich), and c-Myc (9E10, sc-40, IB 1:2,000; Santa Cruz Biotechnology) were used and purchased as indicated.

Techniques: Phospho-proteomics, Residue, Competitive Binding Assay, Expressing, Incubation, Western Blot, Immunoprecipitation, Magnetic Beads, Immunofluorescence, Control, Staining

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: Immunoblotting analysis of immunoprecipitated samples from HEK293T cells co-expressing GFP-STIL with several constructs, such as Myc-Plk4 or GFP-Cep131 a , Myc-Plk4 with GFP-Cep131 (WT or 2A) b , and Myc-Plk4-ND (nondegradable; constitutive active form) with GFP-Cep131 (WT or 2A) c . After immunoprecipitation using GFP-magnetic beads, protein levels were detected using the indicated antibodies. HEK293T cells co-expressing HA-Cep131-WT and GFP-STIL-ΔC (truncated C terminus of STIL, 1-831 aa) ( d ) or GFP-STIL-3A (alanine mutants on residue S1015, S1062, and S1070) ( e ) were immunoprecipitated using HA-magnetic beads, and the proteins levels were observed by immunoblotting. f Endogenous STIL extracted from U2OS cells was immunoprecipitated, and total and pulled down proteins were observed by immunoblotting. Quantification of band intensity was measured by ImageJ (NIH, Bethesda, MD, USA) and indicated as ratios in b – f . g Immunoblotting analysis shows protein levels of Cep131. U2OS cells with stable expression of untagged Cep131-WT and 2A containing resistant sequences to siRNA were transfected with siCon or siCep131 to eliminate endogenous Cep131. h Immunofluorescence analysis Plk4 variations (top panel) and STIL (bottom panel) levels at the centriole in U2OS cells transfected as in g . Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. i The graph shows quantification of the fluorescence intensity of Plk4 (left) and STIL (right) at the centriole. More than 50 centrioles were measured for each condition. * P < 0.05 and ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in a – g

Article Snippet: Rabbit polyclonal antibodies against Cep131 (A301-415A, immunofluorescence (IF) 1:100, immunoblotting (IB) 1:2,000; Bethyl Laboratories, Montgomery, TX, USA), STIL (A302-441A, IF 1:100, IB 1:1,000; Bethyl Laboratories) (ab89314; immunoprecipitation (IP) 2 mg/ml; Abcam, Cambridge, UK;), Plk4 (ab1373398, IB 1:1,000, IP 2 mg/ml; Abcam), γ-tubulin (T5192, IF 1:500, IB 1:3,000; Sigma-Aldrich, St. Louis, MO, USA) HA-tag (Y-11, sc-805, IF 1:100, IB 1:2,000; Santa Cruz Biotechnology; Dallas, TX, USA), and GFP-tag (FL, sc-8334, IB 1:2,000; Santa Cruz Biotechnology), as well as mouse monoclonal antibodies against centrin (20H5, 04-1624, IF 1:100; EMD Millipore, Billerica, MA, USA), SAS-6 (91.390.21, sc-81431, IF 1:100; Santa Cruz Biotechnology), ninein (clone 79-160, MABT29, IF 1:200; EMD Millipore), γ-tubulin (T6557, IF 1:500, IB 1:3,000; Sigma-Aldrich), and c-Myc (9E10, sc-40, IB 1:2,000; Santa Cruz Biotechnology) were used and purchased as indicated.

Techniques: Western Blot, Immunoprecipitation, Expressing, Construct, Magnetic Beads, Residue, Transfection, Immunofluorescence, Fluorescence, Staining, Control

Journal: Cell Death & Disease

Article Title: Cep131 overexpression promotes centrosome amplification and colon cancer progression by regulating Plk4 stability

doi: 10.1038/s41419-019-1778-8

Figure Lengend Snippet: a , b HEK293T cells co-expressing Myc-Plk4 and HA-Ub and/or GFP-Cep131 were treated with MG132 for 6 h and immunoprecipitated. Ubiquitylation properties were analyzed by immunoblotting. c U2OS cells with doxycycline-inducible expression of Myc-Plk4 were treated with 10 μM cycloheximide (CHX) for 8 h. Every 2 h, cells were harvested and analyzed by immunoblotting. d Quantification of Plk4 band intensity measured with ImageJ. Error bars represent means ± SD from three independent experiments. ** P < 0.01, unpaired Student’s t test. NS, not significant. e HEK293T cells stably expressing shGL2 (control) or shSTIL were transfected with HA-Cep131-WT or 2A and immunoprecipitated. Ubiquitylation properties were analyzed by immunoblotting. f Immunofluorescence analysis of excessive accumulations of Plk4 (top panel) and STIL (bottom panel) at the centriole in U2OS cells transfected with Cep131-WT or 2A. Insets are approximately fivefold magnified at the centrosomal region. Scale bar, 10 μm. g Quantification of fluorescence intensity of Cent (left), Plk4 (middle), and STIL (right) at the centriole. Over 50 centrioles were measured for each condition. ** P < 0.01, unpaired Student’s t test. CBB staining use as a loading control in a , b , c , e

Article Snippet: Rabbit polyclonal antibodies against Cep131 (A301-415A, immunofluorescence (IF) 1:100, immunoblotting (IB) 1:2,000; Bethyl Laboratories, Montgomery, TX, USA), STIL (A302-441A, IF 1:100, IB 1:1,000; Bethyl Laboratories) (ab89314; immunoprecipitation (IP) 2 mg/ml; Abcam, Cambridge, UK;), Plk4 (ab1373398, IB 1:1,000, IP 2 mg/ml; Abcam), γ-tubulin (T5192, IF 1:500, IB 1:3,000; Sigma-Aldrich, St. Louis, MO, USA) HA-tag (Y-11, sc-805, IF 1:100, IB 1:2,000; Santa Cruz Biotechnology; Dallas, TX, USA), and GFP-tag (FL, sc-8334, IB 1:2,000; Santa Cruz Biotechnology), as well as mouse monoclonal antibodies against centrin (20H5, 04-1624, IF 1:100; EMD Millipore, Billerica, MA, USA), SAS-6 (91.390.21, sc-81431, IF 1:100; Santa Cruz Biotechnology), ninein (clone 79-160, MABT29, IF 1:200; EMD Millipore), γ-tubulin (T6557, IF 1:500, IB 1:3,000; Sigma-Aldrich), and c-Myc (9E10, sc-40, IB 1:2,000; Santa Cruz Biotechnology) were used and purchased as indicated.

Techniques: Expressing, Immunoprecipitation, Western Blot, Stable Transfection, Control, Transfection, Immunofluorescence, Fluorescence, Staining

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: PP2AB56 promotes the timing of anaphase and mitotic exit. (A–C) PP2AB56 depletion delays anaphase onset. (A,B) RPE1 cells expressing RNAi-resistant B56α and/or H2B–mCherry were transfected with siRNA targeting B56α, B56β, B56γ, B56δ and B56ε (siB56α-ε) or control siRNA (siCtrl) and imaged live. (A) The duration of mitosis was measured for individual cells. Plotted is the fraction of cells that entered anaphase as a function of time in mitosis. (B) Representative micrographs. Numbers indicate time (min) relative to nuclear envelope breakdown (NEBD). The time to metaphase (blue bar) and time from metaphase to anaphase (green bar) is indicated. (C) RPE1 cells expressing H2B–mCherry were treated with siCtrl, siB56α-ε, or a mixture of siRNAs against B56α, B56γ, B56δ and B56ε (siB56) and imaged live. The cumulative anaphase onset is plotted. (D) Depletion of PP2AB56 delays mitotic exit. RPE1 cells were treated with siCtrl, siB56α-ε or mixtures in which an siRNA against a single B56 isoform had been omitted. Cells were treated with nocodazole for 2 h and then imaged live before and after reversine addition. The median time of mitotic exit (line) and exit time of individual cells (open circles) is plotted. Triangles indicate cells that remained in mitosis at the end of the imaging period. (E) siCtrl and siB56 mitotic HeLa lysates were probed by western blotting. Asterisk indicates non-specific band. (F,G) Cyclin B1 proteolysis is delayed in siB56 cells. (F) RPE1 CCNB1Venus/+ siCtrl or siB56 cells were treated as described in D and imaged live. Still images from differential interference contrast (DIC) and fluorescent imaging are shown. Numbers indicate time (min) relative to addition of reversine. (G) Plotted is the fluorescence intensity relative to reversine addition. Each line indicates a single cell and the last time point plotted is either mitotic exit or the experimental end-point (150 min). (H,I) siB56 cells have increased recruitment of Mad2 and BubR1 when Mps1 is inhibited. RPE1 siCtrl or siB56 cells were incubated with nocodazole and MG132, and treated with or without reversine before processing for immunofluorescence microscopy. (H) Maximum intensity projection images of representative cells used for quantification shown in I. (I) Quantification of kinetochore recruitment. Each circle represents the average kinetochore signal of one cell. Line indicates mean; a.u., arbitrary units; n.s., not significant (P>0.05), **P<0.005, ***P<0.0005, Student's two-tailed t-test. Results are representative of three independent experiments. n indicates number of cells analyzed from three experiments (A,C) or single experiment (D). Scale bars: 5 μm.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Expressing, Transfection, Imaging, Western Blot, Fluorescence, Incubation, Immunofluorescence, Microscopy, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: PP2AB56-dependent stimulation of APC/CCdc20 assembly does not require direct binding between B56 and BubR1. (A) BubR1 association with APC/C is reduced in siB56 cells. Mitotic siB56 or siCtrl HeLa cells were used for BubR1 (lanes 4,5) or control (lane 3) IgG IPs and analyzed by western blotting. (B) Quantification of three replicate experiments as in (A). (C–H) Deletion of the KARD in BubR1 does not alter APC/C–Cdc20 association. (C) Schematic for BubR1 depletion and rescue in HeLa cells. (D) BubR1-rescue lysates were analyzed alongside lysates from untreated cells by quantitative western blotting. Endogenous BubR1 signals were normalized to the actin signal, and the value in the untreated sample was set to 1. Solid line indicates intervening lanes have been cropped. (E–H) Lysates (lanes 1,2) and IPs (lanes 3-5) of GFP (E) or Cdc20 (G) were analyzed by western blotting. The experiments in E,G were performed three times and the quantifications are shown in F and H, respectively. (I,J) Comparison of mitotic exit delay in siB56 cells versus BubR1-rescue cells. siCtrl and siB56 cells (I) or BubR1-rescue cells (J) were incubated in nocodazole and reversine and imaged live. Plotted is the fraction of cells that exited mitosis as a function of time after mitotic entry. n, total number of cells analyzed from three independent experiments. Bars are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P<0.005, ***P<0.0005, Student's two-tailed t-test.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Binding Assay, Western Blot, Incubation, Two Tailed Test

Journal: Journal of Cell Science

Article Title: The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis

doi: 10.1242/jcs.201608

Figure Lengend Snippet: B56α interacts with the APC/C constitutively and independently of BubR1. (A,B) B56α co-precipitates with APC/C. (A) Mitotic (mit.) or asynchronous (asy.) HeLa cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting for the indicated proteins. (B) Quantification of three replicate experiments as described in A. Values were normalized to those for the PP2A catalytic subunit (PP2A cat.). (C,D) Mitotic interaction of B56α and APC/C does not require the KARD of BubR1. BubR1 depletion and rescue was performed as described in Fig. 4C, and cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting. (D) Quantification of three replicate experiments as described in C. (E,F) An LxxIxE motif mediates association of APC/C with B56α. Expression of YFP–B56α WT or an R222E mutant was induced by doxycycline in HeLa FLP-In lines, and mitotic cells were collected. (E) IPs for GFP (for YFP-tagged constructs; lanes 4,5) and control (lane 3) IPs were analyzed by western blotting. Asterisk indicates non-specific band. (F) Quantification of three replicate experiments described in E. Data are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P< 0.005, ***P<0.0005, ****P<0.00005, Student's two-tailed t-test.

Article Snippet: Commercial antibodies used in this study include those against: α-tubulin (mouse DM1α–FITC conjugate; Sigma, F2168); β-actin (Sigma, A5316); APC1 (Bethyl Laboratories, A301-653A); APC3 (BD Biosciences, 610455, western blot only); APC7 (Bethyl Laboratories, A302-551A); APC8 (Bethyl Laboratories, A301-182A); BubR1 (BD Transduction Laboratories, 612503; immunofluorescence and western blot only); PP2A catalytic subunit (BD Transduction Laboratories, 610555); PP2A scaffold (Santa Cruz Biotechnology, sc-6112); Mad2 (Bethyl Laboratories, A300-301A); B56γ (Bethyl Laboratories, A303-814A); B56δ (Bethyl Laboratories, A301-098A); and human CREST antiserum (Immunovision, Springsdale, AR, HCT-0100; 1:5000).

Techniques: Western Blot, Expressing, Mutagenesis, Construct, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: APPL1 is a part of the β-catenin-Reptin-HDAC1/2 complex. A , HEK293 cell lysates were subjected to a coimmunoprecipitation assay ( IP ) using anti-Reptin or control IgG, and the resultant precipitates were subjected to immunoblotting ( IB ) with the indicated antibodies. TCL , total cell lysate. B , a 2D BN/SDS-PAGE analysis of HEK293 cell extracts. Multiprotein complexes were resolved on 6–13% acrylamide gradient gel, followed by Tris-glycine-SDS-PAGE (8% gel). Immunoblotting was performed with specific antibodies recognizing the indicated proteins.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Overexpression of APPL proteins reduces the amounts of HDACs bound to Reptin. A , HEK293 cells were transiently transfected with APPL1-Myc and APPL2-Myc. Forty-eight hours post-transfection, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with either anti-Reptin or control IgG. The resultant precipitates were analyzed by immunoblotting ( IB ) against the indicated antibodies ( A ) or were subjected to fluorometric assays for measuring the respective HDAC activities ( C ). Each HDAC activity assay was performed in duplicate, and relative fluorescence unit ( RFU ) values shown are the averages ± S.D. from which the RFU values of assay buffer were subtracted. B , HEK293 cells were cotransfected with plasmids encoding YFP-tagged APPL1 or empty vector. Cell extracts were prepared and immunoprecipitated using anti-HDAC2. Bound proteins were separated and visualized by immunoblotting using anti-HDAC2, anti-HDAC1, anti-Reptin, and anti-APPL1 antibodies.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, HDAC Activity Assay, Fluorescence, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Recruitment of β-catenin, Reptin, and HDAC1 to the promoters of Wnt target genes is affected upon overexpression of APPL proteins. A , overexpression of APPL proteins reduces the amount of Reptin bound to β-catenin. HEK293 cells were transiently transfected with APPL1 or APPL2. Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h. Subsequently, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with anti-β-catenin or control IgG. The resulting precipitates were analyzed by immunoblotting ( IB ) with the indicated antibodies. B , APPL proteins increase the recruitment of β-catenin and reduce the amounts of Reptin and HDAC1 at the Wnt target gene promoters. HEK293 cells were transfected with either empty pcDNA 3.1 ( lane 1 ) or plasmids expressing APPL1 ( lane 2 ) and APPL2 ( lane 3 ). Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h and then subjected to chromatin immunoprecipitation using anti-β-catenin, anti-HDAC1, and anti-Reptin antibodies. PCR was performed from these immunoprecipitates by using the primer pair covering the β-catenin binding sites at the promoters of cyclin D1 and Axin2. PCR products were resolved by agarose gel and stained with ethidium bromide. Lane c , PCR mixture without template.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Staining

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 1 | Design strategies for endocytosis-triggering EndoTags. a, Schema of designed endocytosis mechanisms. Top, design of binding to constitutively cycling receptors at sites that do not overlap with binding sites for natural ligands to avoid competition. Middle, design of binders that trigger endocytosis by eliciting conformational changes in the receptor. The EndoTag binds at two distinct epitopes on the target and actively triggers the conformational change. Bottom, designed endocytosis via receptor clustering. The multivalent EndoTag clusters multiple copies of the target receptor and induces endocytosis. b, Design strategy for sortilin48 and TfR49 EndoTags. c, Cellular uptake of 100 nM AF647-labelled Sort_EndoTags, TfR-EndoTags or LHDB22 scaffold control for 2 h in U-251MG cells. Data were normalized to the 100 nM AF647-labelled LHDB group (no endocytosis). MFI, mean fluorescence intensity. d, Confocal imaging of Sort_EndoTag (red) and lysosomal marker (green, AF488-labelled LysoTracker) after 24 h incubation in U-251MG cells. e, Design strategy for

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Binding Assay, Control, Fluorescence, Imaging, Marker, Incubation

Journal: Nature

Article Title: Designed endocytosis-inducing proteins degrade targets and amplify signals.

doi: 10.1038/s41586-024-07948-2

Figure Lengend Snippet: Fig. 3 | Clearance of soluble proteins by IGF2R pLYTACs. a, Schema for the use of soluble pLYTACs with IGF_EndoTags. b, Cellular uptake of LHDB–AF647 via LHDA–IGF_EndoTags in Jurkat cells. Cells were incubated with 33 nM LHDB– AF647 with or without 1 μM LHDA–IGF_EndoTags for 24 h, washed twice with cold PBS and analysed by flow cytometry. c, Remaining supernatant LHDB– AF647 levels in Jurkat cells. Jurkat cells were incubated with 100 nM LHDB– AF647 with or without 500 nM LHDA–IGF_EndoTags. At timepoints 24 h and 48 h, the cells were pelleted down, and IgG in the supernatant was quantified using a Neo2 plate reader. IgG level was normalized to the IgG-alone control group. d, Cellular uptake of IgG–AF647 via protein G–IGF_EndoTags in K562 cells. Cells were incubated with 33 nM IgG–AF647 with or without 1 μM protein G–IGF_EndoTag3 for 24 h, washed twice with cold PBS and analysed by flow cytometry. The fold change in MFI was calculated by normalizing to the

Article Snippet: Anti-LAMP2A antibody (Abcam ab18528), goat anti-rabbit- IgG Alexa FluorTM 488 secondary antibody (Thermo Fisher A-11034), anti PD-L1 (sc-518027), anti beta-actin (sc-47778), goat anti-mouse IgG H&L (HRP) (Abcam, ab205719); 800CW goat-anti-mouse or goatanti-rabbit (LI-COR 926-32211), rabbit anti-CTLA4 E1V6T Cell Signaling Technologies (96399) Validation Alexa Fluor® 647 anti-human EGFR Antibody (Biolegend, 352918): Verified Reactivity to Human, FC - Quality tested PE anti-human CD222 (IGF2R) Recombinant Antibody (Biolegend, 364204): Verified Reactivity to Human, ICFC, FC - Quality tested EGFR Monoclonal Antibody (clone 199.12, Invitrogen, AHR5072): Target Species: Human, applications for Immunocytochemistry, Immunofluorescence, Immunoprecipitation, Western Blot IGF2R Antibody Alexa647 (clone 2G11, Novus, NB300-514AF647): Reactivity Hu, Mu, Rt, Bv, Pm; Applications WB, ELISA, Flow, ICC/IF, IHC, IP, CyTOF-ready Human PD-L1 Alexa Fluor® 647-conjugated Antibody (Rndsystems, FAB1562R): Species Reactivity Human, Detects human PD-L1/B7H1 in direct ELISAs.

Techniques: Incubation, Flow Cytometry, Control

Journal: Plant Physiology

Article Title: An SNW/SKI-INTERACTING PROTEIN influences endoreduplication and cell growth in Arabidopsis

doi: 10.1093/plphys/kiac415

Figure Lengend Snippet: SUD3 interacts with DA3 in vivo and in vitro. A, The BiFC assays indicate that SUD3 interacts with DA3 in N. benthamiana . nYFP-SUD3 and cYFP-DA3 were coexpressed in leaves of N. benthamiana . Bars =20 μm. B, SUD3 binds DA3 in vitro. DA3 fused to MBP tag was pulled down by His-SUD3 immobilized on His beads and then probed with His and MBP antibodies. C, SUD3 associates with DA3 in Arabidopsis. Total proteins extracted from 35S:GFP and gSUD3-GFP;Col-0 Arabidopsis plants were immunoprecipitated (IP) with GFP-Trap-A beads and then probed with anti-GFP and anti-DA3 antibodies, respectively. IB, immunoblot.

Article Snippet: The immunoprecipitates were separated in 10% (v/v) SDS–polyacrylamide gel and detected by immunoblot analysis with anti-GFP (Abmart) and anti-DA3 antibodies, respectively ( Xu et al., 2016 ).

Techniques: In Vivo, In Vitro, Immunoprecipitation, Western Blot

Journal: Plant Physiology

Article Title: An SNW/SKI-INTERACTING PROTEIN influences endoreduplication and cell growth in Arabidopsis

doi: 10.1093/plphys/kiac415

Figure Lengend Snippet: SUD3 physically interacts with UVI4. A, BiFC assays showing that SUD3 interacts with UVI4 in N. benthamiana . cYFP-UVI4 was coexpressed with nYFP-SUD3 in leaves of N. benthamiana . Bars = 20 μm. B, Interaction between SUD3 and UVI4 in the co-immunoprecipitation assays. Nicotiana benthamiana leaves were transformed by injection of Agrobacterium GV3101 cells harboring Pro35S:Myc-UVI4 and Pro35S:GFP , Pro35S:Myc-UVI4 and Pro35S:GFP-DA3 or Pro35S:Myc-UVI4 and Pro35S:GFP-SUD3 plasmids. Total proteins were immunoprecipitated with GFP-Trap-A, and the immunoblot was probed with anti-GFP and anti-MYC antibodies, respectively. C, SUD3 physically interacts with UVI4 in vitro. GST-UVI4 was incubated with MBP-SUD3 and pulled down by MBP-SUD3 and detected by immunoblot with anti-GST antibody.

Article Snippet: The immunoprecipitates were separated in 10% (v/v) SDS–polyacrylamide gel and detected by immunoblot analysis with anti-GFP (Abmart) and anti-DA3 antibodies, respectively ( Xu et al., 2016 ).

Techniques: Immunoprecipitation, Transformation Assay, Injection, Western Blot, In Vitro, Incubation