Fig. 7. Influence of S20 modification on CHIP post-translational half-life.

a NRCMs infected with AdV-Flag or AdV-Flag-PDE5 to reduce PKG activation, and with SIL added to enhance PKG activation placed in media containing cycloheximide (CHX, 100 µM) to block protein synthesis and H₂O₂ (50 µM) to induce oxidative stress. N = 4–5 separate experiments with 5 independent time points per experiment. ANCOVA results for differences between paired relations: *p = 0.001; ^†0.000009. b Same general protocol but with NRCMs over-expressing either S20A or S20E CHIP mutants. P-value for slope difference by ANCOVA. c Same protocol but WT and K31A CHIP mutants. P-value for offset difference by ANCOVA. d Hsp70/Hsp40 dependent protein refolding ATPase assay showing capacity of CHIP to reduce activity in a dose dependent manner in wild type (WT), S20A, and S20E CHIP mutants. K31A CHIP expression prevents this negative modulation. N = 9 biological replicants, RMANOVA performed within each experiment, Dunnets mct: *p = 0.004; **0.006, ***0.002; ^†0.0005, ^††0.0002, ^‡p = 0.002 – each versus no CHIP added (blue bar). Source data are provided as a Source Data file. Regressions (a–c) and summary plot (d) each display mean ± SEM.