Anti phospho pka

To obtain protein samples for the experiment, cultured cells and mouse adipose tissue were collected and treated with lysis buffer (PRO-PREP; Intron Biotechnology, Seoul, Republic of Korea) for 60 min at 4°C. The extracted protein samples were between 35 and 40 μg and subjected to SDS-PAGE on gels ranging from 7 to 12%, followed by transfer to nitrocellulose membranes (Amersham Biosciences, Westborough, MA, USA). Then, primary antibodies specific to the target protein were used to probe the membranes, followed by incubation with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, USA). Enhanced chemiluminescence kits (Amersham Biosciences) were used to detect signals.
The following antibodies were used for the experiments: anti-SCD1 (1:1,500), anti-SREBP1 (1:1,500), anti-phospho-PKA (1:1,000), anti-PKA (1:2,000), anti-phospho-p38 (1:1,000), anti-p38 (1:2,000), and anti-β-actin (1:2,500) from Santa Cruz Biotechnology and anti-phospho hormonesensitive lipase (HSL) (1:1,000) and anti-HSL (1:2,000) from Cell Signaling Technology (Danvers, MA, USA).

After the stimulation cells at confluence were lysed in ice with complete tablet buffer (Roche) supplemented with 2 m M sodium orthovanadate, 30 μg proteins from each lysate were loaded onto 10 or 5% SDS-PAGE gels, and PVDF (polyvinylidene difluoride) membranes (GE Healthcare Europe GmbH; Milan, Italy) were incubated overnight at 4 ° C with anti-KI67 (1: 500; Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) (p-ERK) (1: 1,000; Euroclone, Milan, Italy), anti-p44/42 MAPK (ERK1/2) (1: 1,000; Euroclone), anti-M1 receptor (1: 250; Santa Cruz, CA, USA), anti-phospho-PKA (1: 250; Santa Cruz, CA, USA), and anti PKC (1: 250; Santa Cruz, CA, USA). Protein expression was normalized to the specific total protein (if possible) and verified through β-actin detection (1: 5,000; Sigma-Aldrich) and expressed as a mean ± SD (%).