Western blot was performed as described previously (31 (link)). Briefly, equal protein samples were loaded into a 4–20% HCL gel, transferred to a nitrocellulose membrane, and incubated overnight in 1:500–1:1,000 antibody dilutions in 5% milk with Tris-buffered saline with Tween for phosphorylated (p)ATser473, total AKT, pFOXO1ser256, total FOXO1, pERKthr202/thr204 (Cell Signaling Technology), pIRtyr972 (Invitrogen), and total ERK (Santa Cruz). Image J software was used to quantify mean intensity of equal-area sections representing each sample.
Pirtyr972
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PIRtyr972 is a laboratory equipment product from Thermo Fisher Scientific. It is designed to perform specific functions within a laboratory setting, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Total erk, by Santa Cruz Biotechnology (1 mentions)
P erk thr202 thr204, by Cell Signaling Technology (1 mentions)
Total foxo1, by Cell Signaling Technology (1 mentions)
P foxo1 ser256, by Cell Signaling Technology (1 mentions)
Proteinase and phosphatase inhibitor, by Roche (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Caveolin 3, by BD (1 mentions)
Caveolin 2, by BD (1 mentions)
Gapdh abs16, by Merck Group (1 mentions)
Mabt523, by Merck Group (1 mentions)
Caveolin 1, by BD (1 mentions)
2 protocols using pirtyr972
1
Quantitative Western Blot Analysis
2
Protein Sample Preparation and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
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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).
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