Protein samples were prepared as previously described.
10 (link),
13 (link),
14 (link),
31 (link),
32 (link) Ventricular tissue fragments were disrupted with a PYREX® Potter‐Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The homogenate was centrifuged at 15,000 g at 4°C for 15 min, and the supernatant was saved for immunoblotting. Total protein extracts were run in equal protein amount on SDS‐PAGE electrophoresis. The blots were probed with primary antibodies to ROCK1 (sc‐5560), ROCK2 (sc‐5561), Gαq (E‐17) (sc‐393), β1‐adrenergic receptor (β1‐AR) (sc‐568), adenylyl cyclase V/VI (AC5/6) (sc‐590) from Santa Cruz Biotechnology (Dallas, TX, USA), caveolin‐1 (#610407), caveolin‐2 (#610685), caveolin‐3 (#610421) from BD Biosciences (San Jose, CA, USA), ROCK1 (#4035), insulin receptor (IR) β (#3025), p‐Akt‐Ser473 (#9271), p‐Akt‐Thr308 (#9275), Akt (#9272), p‐GSK‐3β‐Ser9 (#9336) from Cell Signaling Technology (Danvers, MA, USA), p‐IR‐Tyr972 (#44800G) from Invitrogen. All blots were normalized to GAPDH (ABS16; MilliporeSigma, Burlington, MA, USA) or to actin (MABT523; MilliporeSigma).
10 (link),
13 (link),
14 (link),
31 (link),
32 (link) Ventricular tissue fragments were disrupted with a PYREX® Potter‐Elvehjem tissue grinder on ice in lysis buffer containing proteinase and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The homogenate was centrifuged at 15,000 g at 4°C for 15 min, and the supernatant was saved for immunoblotting. Total protein extracts were run in equal protein amount on SDS‐PAGE electrophoresis. The blots were probed with primary antibodies to ROCK1 (sc‐5560), ROCK2 (sc‐5561), Gαq (E‐17) (sc‐393), β1‐adrenergic receptor (β1‐AR) (sc‐568), adenylyl cyclase V/VI (AC5/6) (sc‐590) from Santa Cruz Biotechnology (Dallas, TX, USA), caveolin‐1 (#610407), caveolin‐2 (#610685), caveolin‐3 (#610421) from BD Biosciences (San Jose, CA, USA), ROCK1 (#4035), insulin receptor (IR) β (#3025), p‐Akt‐Ser473 (#9271), p‐Akt‐Thr308 (#9275), Akt (#9272), p‐GSK‐3β‐Ser9 (#9336) from Cell Signaling Technology (Danvers, MA, USA), p‐IR‐Tyr972 (#44800G) from Invitrogen. All blots were normalized to GAPDH (ABS16; MilliporeSigma, Burlington, MA, USA) or to actin (MABT523; MilliporeSigma).
