Fig. 1: Effect of simulated ischemia (SI)+/− PKG blockade on ubiquitinated proteins and CHIP.
a Left: example immunoblot for ubiquitinated proteins from myocytes infected with adenovirus (AdV) for Flag or PDE5A-Flag (PDE5), the latter to suppress PKG activation (c.f. Supplementary Fig. 1a, b). Cells are then subjected to normal conditions or SI. Right: summary results, n = 6 biological replicates/group, 2WANOVA, Tukey multiple comparisons test (mct): *p < 10−8, †p = 0.0014, ‡p = 4 × 10−6. b Blocking PKG activation by PDE5Aoe reduces proteasome activity similarly at baseline or after SI (n = 6 biological replicates/group, 2WANOVA, Tukey mct: *p = 8 × 10−6, †8 × 10−7, ‡1 × 10−4, §0.002). c CHIP protein expression increases after SI but this is blocked by PDE5Aoe, 6 biological replicates/group, 2WANOVA, Tukey mct, *0.001, †7 × 10−6, ‡0.015. d CHIP mRNA expression is unaltered between groups, six biological replicates/group; 1WANOVA, p = 0.7. e Immunoblot showing efficacy of CHIP protein suppression in basal and SI conditions after incubation with specific but not scrambled (Scr) silencing RNA (siRNA), summary in Supplementary Fig. 1c. f LDH/MTT ratio in NRCMs exposed to control or SI conditions, +/−siRNA to CHIP (100 pM) or Scr, and with or without PDE5A inhibitor, sildenafil (SIL, 1 µM). n = 6 biological replicates/group; 1WANOVA, Sidak mct: *p = 9 × 10−6, †0.002, ‡1.2 × 10−9, §5 × 10−5. Source data are provided as a Source Data file. Individual data and mean ± SEM shown in each summary panel.
