Anti gsk3β ser9

CK2α antisera was prepared as described^{44 (link)}, anti-p-Akt (Ser129) (catalog ab133458), anti-CK2β (catalog ab76025), anti-GLUT4 used for Wb (catalog ab62375) and anti-VAMP2 (catalog ab3347) antibodies were from Abcam (Cambridge, UK), anti-β-actin (catalog A5441) from Sigma-Aldrich. Antibodies raised against PTEN (catalog sc-7974), Akt1/2/3 (catalog sc-8312) Na/K ATPase (catalog sc-28800) and GLUT4 (catalog sc-1606) used for immunolocalization were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while anti-phospho-PTEN (Ser370) (catalog 07–889) was from Merck Millipore (Darmstadt, Germany). Anti-p-Akt (Thr308) (catalog #13038), anti-p-Akt (Ser473) (catalog #4060), anti-AS160 (catalog #2670), anti-p-AS160 (Thr642) (catalog #4288), anti-p-PRAS40 (Thr246) (catalog #13175), anti-GSK3β (Ser9) (catalog #5558), anti-p-FoxO1 (Ser253) (catalog #9461), anti-FoxO1 (catalog #2880) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

The phosphorylation assay was determined by western blot assay. Cultures were serum-starved for 3 h before the herbal or activator application. After drug treatment, including all activators, the cultures were collected immediately in lysis buffer, and the protein were subjected to SDS-PAGE analysis. After transferring the proteins to membranes, the membranes were incubated with anti-GSK 3β (Ser 9) (Cell Signaling) and anti-ERα (Ser 118) (Cell Signaling) at 4°C for 12 h. Following incubation in HRP-conjugated anti-rabbit secondary antibodies in 1: 5,000 dilutions for 3 h at room temperature, the immune-complexes were visualized by the ECL method (Amersham Biosciences). The band intensities in the control and agonist-stimulated samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer, using in each case a calibration plot constructed from a parallel gel with serial dilutions of one of the samples.

Total proteins isolated from LV tissues were rapidly minced and homogenized in 1 × RIPA ice‐cold lysis buffer (with protease inhibitor). After centrifuging at 800 g for 5 min. at 4°C to remove nuclei, the supernatant was further centrifuged at 12,000 g for 30 min. to obtain the mitochondrial pellets and the cytosolic extracts (supernatant). Equal amount of mitochondrial fractions or cytosolic proteins was separated in 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore). The membranes were immunoblotted with anti‐Drp1Ser⁶¹⁶, anti‐caspase9, anti‐PKC‐δ, anti‐PKC‐ε, anti‐GSK‐3β and anti‐GSK‐3β^{Ser 9} (Cell Signaling, Beverly, MA, USA) at 4°C overnight. After washing by 0.1%PBS for three times, the blots were incubated with HRP‐conjugated anti‐IgG for 2 hrs. Immunoreactivities were detected using the enhanced chemiluminescence reaction system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Densitometric analysis was performed using QuantityOne software version 4.5.2 (Bio‐Rad, Hercules, CA, USA). In brief, the density area of each band can be automatically identified and outlined by the software, and then the brightness value for each band was obtained. The ratio of each detected protein to β‐actin represented to their relative protein levels.