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