Journal: Gut Pathogens

Article Title: CMTM3 protects the gastric epithelial cells from apoptosis and promotes IL-8 by stabilizing NEMO during Helicobacter pylori infection

doi: 10.1186/s13099-023-00533-4

Figure Lengend Snippet: CMTM3 reduces NEMO degradation. a The immunoprecipitation analysis of GES-1 cells. Cells expressing pCMV-NEMO-3Flag were immunoprecipitated by an anti-Flag antibody and immunoblotted by anti-NEMO and anti-CMTM3 antibodies. b Co-localization of CMTM3 and NEMO in GES-1 cells was investigated by immunofluorescence co-localization analysis after H. pylori infection for 24 h. Scale bar, 10 μm. Hochest was used for nuclear staining. c, d CMTM3 knockout GES-1 cells were treated with cycloheximide (25 µg/mL) and proteins were subjected to immunoblotting (c) . Data is representative of three independent experiments. The remaining NEMO was quantified (d) . e The ubiquitylation of NEMO. HEK 293 T cells were transfected with the indicated vectors. After 48 h transfection, cells were treated with MG132 (10 μM) for 6 h. Then cells were lysed and immunoprecipitated with anti-Flag antibody and analyzed by immunoblotting with the indicated antibodies. f CMTM3 KO GES-1 cells were treated with MG132 (10 μM) and collected at indicated time points. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. g The expression the phosphorylated IKBα (p-IKBα) and phosphorylated p65 (p-p65) were analyzed in CMTM3 KO GES-1 cells.*p < 0.05

Article Snippet: The cells were collected and lysed by immunoprecipitation RIPA buffer (Solarbio) with protease and phosphatase inhibitors, on ice for 30 min.

Techniques: Immunoprecipitation, Expressing, Immunofluorescence, Infection, Staining, Knock-Out, Western Blot, Transfection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Establishment of a novel method to assess MEK1/2 inhibition in PBMCs for clinical drug development

doi: 10.3389/fcell.2022.1063692

Figure Lengend Snippet: Comparable results for sample analysis with Wes™ and MSD system. (A,B) Comparison of cell lysis using 1x RIPA or Complete lysis buffer as preliminary step for Wes™ and MSD system comparison. Cell treatment was performed as previously described and cells were lysed using either 1x RIPA (dark grey) or Complete lysis buffer (light grey). The ERK1/2 phosphorylation status was assessed with the Wes™ system. Two-way ANOVA with Šídák correction was used for statistical analysis to compare the results obtained for cell lysis using 1x RIPA or Complete lysis buffer. ns, not statistically significant. N = 3 blood donors. (C,D) Comparison of (C) pERK/ERK (ratio of phosphorylated ERK1/2 to total ERK1/2) and (D) %MEK1/2 inhibition upon sample analysis with the Wes™ and the MSD system. Cell treatment and lysis was performed as previously described and the samples were analyzed with the Wes™ (dark grey bars, blue datapoints) and the MSD system (light grey bars, green datapoints). Two-way ANOVA with Šídák correction was used for statistical analysis to compare the results obtained with Wes™ and MSD platform. ns, not statistically significant. N = 5 blood donors. Data are displayed as (A,C) ratio pERK/ERK and (B,D) %MEK1/2 inhibition compared to DMSO. Data are presented as individual values and mean with standard deviation. Each datapoint represents one blood donor. (E,F) Presentation of pERK1/2 and ERK1/2 levels as Western blot-like bands of one representative blood donor included in (A,B) or (C,D) , respectively. The Western blot-like band data of all blood donors is shown in the .

Article Snippet: • 1x radio-immunoprecipitation assay (RIPA) buffer (used for sample preparation for the WesTM system): 0.24% (w/v) tris base, 0.88% (w/v) NaCl, 0.2% (v/v) 500 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS (Carl Roth), 10% (v/v) glycerol, 0.05% PMSF, 0.01% Benzonase ® Nuclease, protease inhibitor cocktail, and phosphatase inhibitor cocktail in water (ddH 2 O) • Complete lysis buffer (used for sample preparation for the MSD system; use with the WesTM system possible): Inhibitor pack Meso Scale Discovery, 2 mM PMSF Sigma-Aldrich, and 0.1% SDS (Gibco) in Tris lysis buffer (Meso Scale Discovery)

Techniques: Comparison, Lysis, Phospho-proteomics, Inhibition, Standard Deviation, Western Blot