Fig. 3. Functional regulation of UPS by Chip S20 is revealed by gain and loss of function phospho-mutants.
a Upper: representative Western blot of ubiquitinated proteins in NRCMs infected with adenovirus expressing WT, S20E (SE), or S20A (SA) CHIP, and exposed to normal or SI conditions for 48 h; Lower: summary results, data normalized for protein loading, and to control WT-CHIP; three gels, six biological replicates, 2WANOVA, Tukey mct: *p = 7 × 10−8, †3 × 10−5, ‡10−9, §4 × 10−8, ¶<10−9, #0.03. b Cytotoxicity (LDH/MTT assays) from SI is reduced by (SE) and increased by SA CHIP expression versus WT. PDE5A inhibitor sildenafil (SIL) reverses cytotoxicity in WT, but less so with SE or SA expression. 2WANOVA, 1.3 × 10−5 genotype condition interaction; Sidak mct: †p = 1.3 × 10−8 vs SE-SI, 1.2 × 10−9 vs SA-SI; #10−11 SE-SI; ‡<10−11 vs WT-SI + SIL and SE-SI+SIL; *p < 2 × 10−5 vs SI for each respective CHIP group. c Same experiment using AdV expressing combined (S20E, S24E; SE) or (S20A, S24A; SA). 2WANOVA, Sidak mct: †p = 0.01vs SE-SI, 2 × 10−5 vs SA-SI; #4.8 × 10−9 SE-SI; ‡0.03 vs SE-SI+SIL; p = 0.005 vs WT-SI + SIL; *1.4 × 10−5 vs WT-SI, 5.6 × 10−8, vs SA-SI. d Proteasome activity in resting NRCMs is augmented by PKG activation by PDE5 inhibitor sildenafil (SIL) in control myocytes and cells subjected to SI. Results of 2W-ANOVA for SIL effect and for interaction between SIL effect and gene-type of CHIP expressed are provided in the figures. N = 6 biological replicates for all conditions/groups. Source data are provided as a Source Data file. Individual data and mean ± SEM shown in each summary panel.