Figure 3. PP1 dephosphorylates BAG3-pS136 in cells.

(A) Illustrative overview of the small molecule and peptide modulator screen to tune PP1 activity in cells. HEK293 cells transiently expressing FLAG-BAG3 were treated as illustrated, lysed, and captured by a single-step affinity enrichment using anti-FLAG beads. Samples were analyzed by immunoblots. Quantification depicts results of three independent experiments with P-values obtained from t tests (two-tailed). Mean is shown with replicates as scatter plot and error bars represent the SD (*P = 0.015, ***P < 0.001). (B) A7r5 smooth muscle cells were incubated with increasing concentrations of Tautomycetin as indicated. Immunoblots of lysate were used to determine the sensitivity of the titrated inhibitor towards BAG3-pS136. Mean is shown as a barplot with replicates as scatter plots, and error bars represent the SD of four independent experiments. Statistical significance between concentrations is determined with t test with Welch’s correction (*P < 0.05). (C) Analysis of phosphorylation-dependent binding of 14-3-3γ to FLAG-BAG3 in A7r5 muscle cells after 20-min modulation of endogenous PP1 before lysis and FLAG-IP enrichment. Results are presented as a barplot, P-values obtained from a t test (***P = 0.0002, ****P < 0.0001). (D) Immunoblots depicting siRNA-mediated knockdown of all PP1 isoforms combined (PP1α/β/γ) or individual PP1 isoforms (PP1α, PP1β, PP1γ) separately. The displayed blots represent the analyzed protein levels of 24 or 48 h after transfection. (E) Quantification of immunoblotted lysates show level changes of PP1α/β/γ and BAG3 (n = 3 or 4). Quantification depicts results of three or four independent experiments with P-values obtained from t tests (two-tailed, paired). Error bars represent the SD.

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