Anti phosphorylated akt s473

Western blotting analysis was conducted following a well-established protocol [7 (link)]. In summary, proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and subsequently transmitted to a PVDF (polyvinylidene fluoride) membrane (Merck KGaA, Darmstadt, Germany) through a wet blotting technique. To prevent non-specific binding, 5% blocking solution made with skimmed milk was applied to the membrane. The membranes were subsequently left to incubate overnight at 4 °C with primary antibodies, including anti-total-AMPK, anti-phosphorylated AMPK-alpha (T172), anti-total- Akt, anti-phosphorylated Akt (S473) (Cell signaling Technology, Danvers, MA, USA), and anti-β-actin (AbFrontier, Seoul, Republic of Korea). Following primary antibody incubation, the membranes were treated with secondary antibodies specific to rabbit IgG coupled with horseradish peroxidase (AbFrontier, Seoul, Republic of Korea). The detection of the bound antibodies was facilitated by the EzWestLumi Plus kit (ATTO Corporation, Tokyo, Japan) and visualized using the LuminoGraph II system (ATTO Corporation, Tokyo, Japan) for chemiluminescence.

Western blotting was carried out as described previously [37 (link)]. The following primary antibodies were used for western blotting: anti-MED23 (BD, 550492), anti-MED16 (Bethyl, A303-668A, RRID: AB_11205632), anti-MED24 (Bethyl, A301-472A, RRID: AB_999675), anti-phosphorylated-Erk1/2 (Thr202/Tyr204) (Santa Cruz, sc-16982), anti-Erk (K-23) (Santa Cruz Biotechnology, sc-94-G, RRID: AB_631456), anti-phosphorylated-Akt (S473) (Cell signaling, 9271), anti-Akt (Cell signaling Technology, 9272, RRID: AB_329827), anti-Ets1 (Santa Cruz, sc-350x), anti-Sprouty2 (Santa Cruz, sc-30049), anti-Src (Cell signaling Technology, 2108s, RRID: AB_331137) and β-Actin (Sigma, A5441).