Akt pan 40d4

Human Skeletal Myoblasts (#A11440 or #A12555), low glucose Dulbecco's Modified Eagle Medium (#11885092), Horse Serum (#26050070), Bovine Serum Albumin (#A9576), and Pierce A/G Magnetic Beads (#88802) were purchased from Thermofisher Scientific Antibodies for Total AKT1 (#2938), Total AKT2 (#2964), Total AKT3 (#14293), Total AKT (#4685), p‐AKT S473 (#4060), p‐AKT1 S473 (#9018), p‐AKT2 S474 (#8599), AKT (Pan) (C67E7) (rabbit monoclonal; #4691), AKT (Pan) (40D4) (mouse monoclonal; #2920), and Human Insulin‐like Growth Factor 1 (#8917) were purchased from Cell Signaling Technologies. Secondary antibodies Anti‐Rabbit IgG HRP‐Linked Antibody (PN#7074) and Anti‐Mouse IgG HRP‐Linked Antibody (PN#7076) were purchased from Cell Signaling Technologies. Human Insulin Solution (#I9278) was purchased from Sigma Aldrich ImageJ was downloaded from the National Institutes of Health website.

Following a four hour fast, a subset of mice was randomized to intraperitoneal injection of 1 U/kg insulin or an equivalent volume of saline as a control. After 15 min, the mice were euthanized with CO₂ [64 (link),65 (link)]. Liver, skeletal muscle, and gonadal fat were preserved in liquid nitrogen for future western blot analysis. Western blot was performed by placing 50 μg of protein lysates in a RIPA buffer + 1% SDS (Bio-Rad) + protease and phosphatase inhibitors (CST), followed by Pierce BCA protein quantitation (ThermoScientific, Waltham, MA, USA), resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membrane as previously described [66 (link)]. The primary antibodies utilized were: Phospho-Akt (S473) (Cell Signaling Technologies, 4060S), Akt-pan (40D4) (Cell Signaling Technologies, 2920S), Phospho-S6 (S240/244), (Cell Signaling Technologies, 5364S), S6 ribosomal protein (Cell Signaling Technologies, 2317S), and Vinculin (Cell Signaling Technologies, 13901S). The blot was visualized with SuperSignal West Pico PLUS (ThermoScientific), as per the manufacturer’s protocol. Densitometry analysis was performed with NIH ImageJ software. Values were normalized to the PBS control group.

Approximately 25–30 mg of tissue was lysed by homogenization and sonication in RIPA buffer (Cell Signaling Technologies), 1% SDS, and protease and phosphatase inhibitor cocktails (Cell Signaling Technologies). Following a Pierce BCA assay (ThermoScientific) to quantify protein, 35–50 μg of protein lysate was resolved on 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat dry milk, and incubated with primary antibodies overnight, prior to treatment with HRP-conjugated secondary antibodies. The primary antibodies used were as follows: phospho-Akt (S473) (Cell Signaling Technologies, 4060S) at 1:1000, Akt-pan (40D4) (Cell Signaling Technologies, 2920S) at 1:500, phospho-S6 (S240/244) (Cell Signaling Technologies, 5364S) at 1:1000, S6 ribosomal protein (Cell Signaling Technologies, 2317S) at 1:1000, MUP (Santa Cruz Biotechnology, sc-166429) at 1:500, and vinculin (Cell Signaling Technologies, 13901S) at 1:1000. The blot was visualized with SuperSignal West Pico PLUS (ThermoScientific), per the manufacturer’s instructions. Densitometry analysis was performed with NIH ImageJ/FIJI software. Values are presented relative to each respective saline control group.