Phospho pka substrate rrxs t

Antibodies directed against AMPKα (1/1000, Rabbit), ACC (1/1000, Rabbit), phospho-Ser⁷⁹ACC (1/1000, Rabbit), phospho-PKA substrate (RRXS*/T*) (1/2000, Rabbit), phospho-AMPK substrate (1/1000, Rabbit), and phospho-Ser¹³³CREB (1/1000, Rabbit) were obtained from Cell Signaling technology (Danvers, MA, USA). Anti phospho-Thr¹⁷²AMPKα (1/1000, Rabbit), CREB (1/500, Rabbit), Arc (1/500, Mouse), c-Fos (1/500, Mouse), and EgrI (1/500, Rabbit) antibodies were from Santa-Cruz (Dallas, TX, USA). Anti-actin (1/15 000, Mouse) antibody was from BD Bioscience (Franklin Lakes, NJ, USA). HRP-coupled secondary antibodies directed against the primary antibodies’ hosts were obtained from Cell Signaling technology. Bicuculline (Bic), H 89, PKI 14-22 amide, NKY 80, SQ 22536, and KH 7 were purchased from Tocris (Bristol, UK), 4-aminopyridine (4-AP) was purchased from Sigma (St Louis, MO, USA), and Compound C (Cc) was from Santa Cruz (Dallas, TX, USA).

Whole-cell protein lysates were extracted with RIPA buffer, subjected to SDS-PAGE, transferred to nitrocellulose membranes using tank blot method and incubated with primary antibodies as previously described [14 (link)]. HRP-coupled secondary antibodies were used and chemiluminescence was detected and quantified using the G:BOX Chemi XX9 system with GeneSys and GeneTools software (SynGene, Bengaluru, Karnataka, India). The following antibodies were used: b-Actin (A1978, Thermo Fisher Scientific), phospho-AKT (Ser473) (#4060, Cell Signalling Technologies, Danvers, MA, USA), AKT (#4691, Cell Signalling Technologies), phospho-PKA substrate (RRXS*/T*) (#9624, Cell Signalling Technologies (CST)), p44/42 MAPK (Erk1/2) (#4695, CST), phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) (#4370, CST), ADIPOQ (#2789, CST), PPARG (#2430, CST), anti-rabbit-HRP (#7074, CST) and anti-mouse-HRP (#7076, CST).