Anti p pkcα

Immunoblotting analysis was performed as already described elsewhere^{40 (link)–42 (link)}. Briefly, the proteins from hearts or cell lysates were homogenized in RIPA buffer (Sigma-Aldrich) containing phosphatase inhibitor (Phos-STOP, Roche) and protease inhibitor (Complete Mini EDTA-free, Roche). Equal amounts of proteins (50 mg) underwent SDS-polyacrylamide gel electrophoresis (PAGE) and were transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies (anti-BNIP3L, anti-fibronectin anti-InsP3R: Santa Cruz; anti-TGF-β, anti-smad2/3, anti-Bcl-XL: CST; anti-collagen I: abcam, anti-PKCα and anti-p-PKCα: abcam) and then probed with horseradish peroxidase conjugated secondary antibodies. Blots were visualized with the use of SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific, USA) and images were captured with an ImageQuant LAS 4000 (GE,USA). The densities of bands were quantified by use of Image-Quant TL software (GE Healthcare).

Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).