Pgsk3 ser 9 21

Typically, 10 µg of protein was resolved by SDS–PAGE, transferred to PVDF membranes and immunoblotted as previously described [5 (link)]. Antibodies detecting TRARG1 (sc-292062 or sc-377025) and 14-3-3 (sc-1657) were from Santa Cruz Biotechnology. Antibodies against pHSL S660 (#4126), pp90RSK (#9344), p4EBP1 S65 (#9456), pTBC1D4 T642 (#4288S), pAKT S473 (#4051), pAKT T308 (#9275L), AKT (#4685), HA (#C29F4), GSK3α (#9338S), GSK3β (#9832S), pGSK3 Ser 9/21 (#8566S), pGS S641 (#3891) and Caveolin1 (#3267) were purchased from Cell Signaling Technology. Anti-α-tubulin (#T9026) was from Sigma–Aldrich. Antibody against TBC1D4 was produced as previously described [4 (link)].

Human PC3, DU145 and LNCaP cell lines were obtained from ATCC (Manassas, VA) and maintained in DMEM High Glucose (PC3 and DU145) or RPMI-1640 (LNCaP) (HyClone) with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO₂ humidified atmosphere at 37°C. Primary antibodies against cleaved caspase-3, cleaved caspase-9, N-Cadherin, p-GSK3 Ser9/21, β-catenin and Snail were purchased from Cell Signaling (Boston, MA), and anti-β-actin was from Sigma (St Louis, MO). Anti-mouse and anti-rabbit HRP conjugated secondary antibodies were obtained from BioRad (Hercules, CA). Human dermal microvascular endothelial cells (HMEC) were obtained from ATCC. Cells were maintained in Endothelial Cell Basal Medium-2 (Lonza) with Microvascular Endothelial Cell Growth Medium-2 Bullet Kit (Lonza). All cultures were maintained in a humidified 5% CO2 incubator at 37°C, and routinely passaged when 80–90% confluent.