Journal: Science Advances

Article Title: Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein

doi: 10.1126/sciadv.adf5143

Figure Lengend Snippet: ( A ) Illustration of the domains of the proteins. Scale bar, 100 amino acid residues. ( B ) Binding of Nck, WISH, and GAS7 isoforms to N-WASP and its ΔPRR mutant. Nck, WISH, and GAS7 isoforms (1 μM) as GST fusion proteins were incubated with N-WASP and its ΔPRR mutant (0.5 μM). The bound proteins were analyzed by SDS–polyacrylamide gel electrophoresis, followed by Western blotting. GST was used as a negative control, and (−) indicates GST fusion protein alone. ( C and D ) Binding curve of immobilized GAS7b (0.1 μM) without GST to N-WASP (C) and WASP and its S339Y, P359T, and P373S mutants (D) by the biolayer interferometry analysis. The K d values ( n = 3) are shown on the right. The means ± SE are shown for the sensorgrams and K d values ( n = 3). ( E ) Binding of N-WASP and GAS7 isoforms to Nck and Grb2 (1 μM) as in (B). MW, molecular weight. ( F ) Binding of WISH and its Y52A mutant to splicing isoforms of GAS7 (0.5 μM) as in (B). ( G ) Binding curve of the immobilized Nck (0.1 μM) to WASP and its S339Y, P359T, and P373S mutants by the biolayer interferometry analysis, as in (D). ( H ) Binding of WISH and its Y52A mutant to N-WASP (0.5 μM) as in (B). ( I ) Binding of WISH and its Y52A mutant to Nck (0.5 μM) as in (B). ( J ) Binding curve of the immobilized WISH (0.1 μM) (H) to WASP and its S339Y, P359T, and P373S mutants by the biolayer interferometry analysis, as in (D). The P values were obtained using the one-way ANOVA with Dunnett’s post hoc analysis (D and G) and the Kruskal-Wallis test, followed by Dunn’s test (J). Significance values are * P < 0.05 and **** P < 0.0001. ns, not significant.

Article Snippet: The membranes were incubated with the primary antibody: mouse anti-GAS7 (clone 2F6, TA501756, OriGene), anti-Grb2 (Sc-8034, OriGene), anti-Nck (SC-20026, Santa Cruz Biotechnology), anti–N-WASP (#4848, Cell Signaling Technology), anti-WASP (#48606, Cell Signaling Technology), anti-SPIN90 (Ab88467, Abcam), anti-mCherry (71615, Cell Signaling Technology), and anti–glyceraldehyde-3-phosphate dehydrogenase (SC16657, Santa Cruz Biotechnology) at a 1:10,000 dilution, followed by an anti-mouse or anti-rabbit IgG alkaline phosphatase conjugate (Promega) secondary antibody in PBS-T at a 1:10,000 dilution.

Techniques: Binding Assay, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Negative Control, Molecular Weight

Journal: Breast Cancer Research : BCR

Article Title: Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

doi: 10.1186/bcr2193

Figure Lengend Snippet: Constitutive HSP90A expression sensitizes cells to apoptosis . (a) Protein extracts were prepared from control parental HC11 or heat shock protein 90 alpha (HSP90α)-HC11 (line 2) cells that were cultured as undifferentiated (U), competent (C) or differentiated (D) cells. (b, c) Differentiated parental HC11 and two different pools of HC11-HSP90α cells (lines 1 and/or 2) were induced to differentiate and then starved of lactogenic hormones and serum for the time indicated. Equal amounts of protein were resolved by SDS-PAGE (15%). An antibody was used to detect phosphorylated-histone 2A.X, and an antibody against GRB2 was used as a loading control. (d) Differentiated control HC11 and HC11-HSP90α cells (line 2) were starved or not apoptosis was assessed by the relative quantities of mono- and oligo-nucleosomes (apoptotic index). Each bar represents the average of three experiments with standard deviation. t-test, *p = 0.02 at 24 hours, **p = 0.00004 at 48 hours.

Article Snippet: Mouse anti-phospho-histone 2A.X (Ser139) clone JBW301 and anti-GRB2 antibodies (Upstate, Charlottesville, USA) and mouse anti-GRB2 (BD Biosciences, Ontario, Canada) were also acquired.

Techniques: Expressing, Cell Culture, SDS Page, Standard Deviation

Journal: bioRxiv

Article Title: p57 Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation

doi: 10.1101/2022.09.09.507138

Figure Lengend Snippet: (A) p57 or p57 CK- was immunoprecipitated with Myc antibodies from HEK293 cells transfected with tGFP-Ascl2 and/or Myc-p57 or Myc-p57 CK- and the presence of Ascl2 was assessed by immunoblotting (n=3). Amounts of transfected proteins in lysates are shown. Grb2 was used as loading control. (B) Ascl2 was immunoprecipitated with tGFP antibodies from HEK293 cells transfected with tGFP-Ascl2 and/or Myc-p57 or Myc-p57 CK- and the presence of p57 was determined by immunoblot (n=3). Amounts of transfected proteins in lysates are shown. Grb2 was used as loading control. (C) Proximity Ligation Assay (PLA) using p57 and Ascl2 antibodies on endogenous proteins in SW480 cells. F-actin was stained with phalloidin and DNA with Hoechst 33342. Scale bars, 50 µm. Representative images from three independent experiments. (D) Schematic of a pZsGreen reporter construct in which a destabilized GFP (½ life ∼ 1 h) is cloned downstream of a promoter to measure promoter activity. (E-F) HEK293 cells were co-transfected with 100 ng of a reporter construct (pZsGreen DA [no promoter, negative control], or pZsGreen pGAPDH [positive control], or pZsGreen pPhox2a [Ascl1 target], or pZsGreen pSox9, or pZsGreen pLgr5 [Ascl2 targets]), 100 ng of an Ascl2 expression vector, and the indicated amounts of a p57 (E) or p57 CK- (F) expression vector. The number of fluorescent cells was quantified using an Incucyte FLR. Scale bars, 200 µm. Graphs represent the mean fluorescent object density normalized to that of GAPDH in the same condition from four independent experiments. Graphs show means ± SEM. ns: p > 0.05; *: p ≤ 0.05; ***: p ≤ 0.001; ****: p ≤ 0.0001. (G) Expression levels FPKM of Ascl2 target genes from in RNA-Seq data from p57 +/+ and p57 KO Hopx-GFP + ISCs from . Graphs show means ± SEM. ns: p > 0.05; *: p ≤ 0.05; ***: p ≤ 0.001.

Article Snippet: Mouse anti-Grb2 (610112) and E-cadherin (610182) were purchased from BD Transduction Laboratories.

Techniques: Immunoprecipitation, Transfection, Western Blot, Proximity Ligation Assay, Staining, Construct, Clone Assay, Activity Assay, Negative Control, Positive Control, Expressing, Plasmid Preparation, RNA Sequencing Assay

Journal: bioRxiv

Article Title: p57 Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation

doi: 10.1101/2022.09.09.507138

Figure Lengend Snippet: (A, B) HDAC7 (A) or mSin3a (B) were immunoprecipitated from HEK293 cells transfected with Flag-HDAC7 or Myc-mSin3a and/or Flag-p57 and/or Flag-p57 CK- and the presence of p57 was assessed by immunoblotting (n=3). Amounts of transfected proteins in lysates are shown. Actin (A) or Grb2 (B) were used as loading control. (C-D) Endogenous HDAC7 (C), mSin3a and p57 (D) were immunoprecipitated from SW480 cells lysates and the presence of endogenous p57, HDAC7 and/or mSin3a was assessed by immunoblotting. Protein A beads alone and control IgG were used as negative control. Respective levels of each protein in lysates are shown. β-actin was used as loading control (n=3). (E) PLA for p57/mSin3a proximity and p57/HDAC7 proximity in SW480 cells using p57, mSin3a and/or HDAC7 antibodies. F-actin was stained with phalloidin and DNA with Hoechst 33342. Scale bars, 50 µm. Representative images of three independent experiments. See also

Article Snippet: Mouse anti-Grb2 (610112) and E-cadherin (610182) were purchased from BD Transduction Laboratories.

Techniques: Immunoprecipitation, Transfection, Western Blot, Negative Control, Staining

Journal: EMBO Reports

Article Title: Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

doi: 10.15252/embr.202153693

Figure Lengend Snippet: Antibodies used for immunofluorescence (IF) and Western blotting (WB) and dilutions.

Article Snippet: Anti‐GRB2 (C7) mouse monoclonal , Santa Cruz Biotechnology , sc‐8034 , 1:100 , 1:500.

Techniques: Immunofluorescence, Western Blot

Journal: bioRxiv

Article Title: The SWELL1-LRRC8 complex regulates endothelial AKT-eNOS-mTOR signaling and vascular function

doi: 10.1101/2020.08.04.236182

Figure Lengend Snippet: A , GRB2 immunoprecipitation from Ad-shSCR and Ad-shSWELL1 transduced HUVECs and immunoblot with SWELL1, Cav1 and GRB2 antibodies. Densitometry values for GRB2 co-immunoprecipitated SWELL1 (SWELL1/GRB2) and GRB2 co-immunoprecipitated Cav1 (Cav1/GRB2). GAPDH serves as loading control for input samples. ( B ) GRB2 immunoprecipitation from Ad-shSCR and Ad-shSWELL1 transduced HUVECs and immunoblot with SWELL1, eNOS, Cav1 and GRB2 antibodies. Insulin-stimulation with 100 nM insulin for 10 minutes. Densitometry values for GRB2 co-immunoprecipitated eNOS (eNOS/GRB2). Representative blots from 3 independent experiments. ( C ) Representative endogenous SWELL1 and eNOS immunofluorescence staining in Ad-shSCR and Ad-shSWELL1 transduced HUVECs. Representative image from 6 independent experiments. ( D-E ) Quantification of SWELL1 ( D , n = 6) and eNOS ( E , n = 6) immunofluorescence staining upon SWELL1 KD. Evidence of SWELL1-eNOS colocalization (C, insets) in plasma membrane ( i ) and perinuclear regions ( ii ). Significance between the indicated groups are calculated using a two-tailed Student’s t-test. P-values are indicated on figures. Error bars represent mean ± s.e.m.

Article Snippet: Purified mouse anti-Grb2 was purchased from BD (610111) and Santa Cruz (#sc-255).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Two Tailed Test