Journal: International Journal of Biological Sciences

Article Title: ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

doi: 10.7150/ijbs.71327

Figure Lengend Snippet: USP11 is phosphorylated by activated ERK1/2 at serine 905. (A and B) MCF-7 cell lysates were immunoprecipitated using anti-USP11 (A) or anti-ERK1/2 antibody (B). The immunoprecipitates were then examined with indicated antibodies. (C) MCF-7 cells were treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated with anti-USP11 antibody, followed by western blotting with anti-p-Ser/Thr antibody and other indicated antibodies. (D) Sequence alignment of the ERK1/2 phosphorylation site within USP11 orthologs from different species. (E) MCF-7 cells were transfected with Flag-USP11 wt , Flag-USP11 S905A , empty vector plasmids for 24 h, and then treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated using anti-Flag antibody, followed by western blotting with anti-phospho-MAPK/CDK substrates antibody and other indicated antibodies.

Article Snippet: UltraSignal ECL (catalog no. 4AW011-100) was purchased from 4A Biotech Co., Ltd. Antibodies information: anti-USP11 (Santa Cruz, catalog no. sc-365528/ Abcam, catalog no. ab109232); anti-p21 (Santa Cruz, catalog no. sc-397/ Cell Signaling Technology, catalog no. 2947S); anti-Flag (MBL, catalog no. M185-3L); anti-Myc (MBL, catalog no. M192-3); anti-HA (MBL, catalog no. M180-3); anti-ERK1/2 (Cell Signaling Technology, catalog no. 4695S); anti-phospho-ERK1/2 (Cell Signaling Technology, catalog no. 4376S); anti-phospho-MAPK/CDK Substrates (Cell Signaling Technology, catalog no. 2325S); anti-phosphoserine/threonine (BD Transduction Laboratories, catalog no. M180-3); anti-GAPDH (COOLRUN Life Science, catalog no. AT0002); Dylight 488 (Thermo Fisher Scientific, catalog no. #35502); Dylight 594 (Thermo Fisher Scientific, catalog no. #35560).

Techniques: Immunoprecipitation, Western Blot, Sequencing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Microglial TNFα orchestrates brain phosphorylation during the sleep period and controls homeostatic sleep

doi: 10.1101/2022.03.24.485623

Figure Lengend Snippet: ( A ) Phosphoregulation of phosphatases and kinases by microglial TNFα. Graphs show percentage of phospho-phosphatases and phosphokinases with at least one changing phosphosite in micTNFα-KO vs CTL comparison at W and S. ( B, C ) Density of phosphosites on ( B ) phosphatases and ( C ) kinases modulated by microglial TNFα at W and S. Graphs show the number of significantly changed phosphosites in micTNFα-KO vs CTL comparison per protein weight. Each dot is one phosphoprotein. Phosphatases are shown in grey and regulatory subunits of phosphoprotein phosphatases (reg sub PPP) in blue. Kinases are color-coded according to the major families: calcium/calmodulin regulated kinases (CAMK, red); CDK, MAPK, GSK3 and CLK kinases families (GMGC, blue); protein kinase A, G and C families (AGC, dark green). ( D ) Kinases with altered activity between controls and micTNF-KOs predicted by robust kinase activity inference (RoKAI). Graph shows the z-score value of each kinase assigned by RoKAI. Kinases are color-coded according to kinase group. ( E, F ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. ( E ) Left , Immunoblots of cortical lysates using antibodies specific to MAPK/CDK and PKC target phosphorylation motifs. Right , Ratio between phosphorylated substrates and loading control normalized to CTL at W and S. ( F ) Left , Immunoblots show signal of phosphorylated MARK activation loop. Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at W and S. ( E, F ) n=5 mice per group. *p<0.05, Mann Whitney test.

Article Snippet: Primary antibodies used were as follows: anti-phospho-MAPK/CDK substrate motif (1:1000, Cell Signaling, 2325), anti-phospho-PKC substrate motif (1:1000, Cell Signaling, 6967), anti-phospho-PKA substrate motif (1:1000, Cell Signaling, 9624), anti-phospho-AKT substrate motif (1:1000, Cell Signaling, 9614), anti-phospho MARK family activation loop (1:1000, Cell Signaling, 4836), anti-vinculin (1:5000, Cell Signaling, 13901) and anti-tubulin (1:10000, Millipore, 05-829).

Techniques: Activity Assay, Western Blot, Activation Assay, MANN-WHITNEY