Ppkcζ

Cardiomyocytes were seeded into coverglass bottom dishes. After washing thrice with PBS, cells were fixed with 4% paraformaldehyde (20 min) and permeabilized using 0.3% Triton X-100 (10 min). Then, cells were washed with PBS for another three times and were blocked with 10% goat serum at room temperature for 1 h. Antibodies against PKC-ζ (diluted 1:50), β-catenin (diluted 1:100), p-PKC-ζ (Santa Cruz, diluted 1:100), or Troponin T (Santa Cruz, diluted 1:100) were used to incubate cells overnight at 4°C. Next, cardiomyocytes were incubated with Alexa Fluor 488- or 594-conjugated anti-rabbit or mouse IgG (H + L) secondary antibody (Proteintech Group, diluted 1:200) at room temperature for 1 h. After nuclear staining by DAPI for 10 min, cardiomyocytes were detected using an ultra-high-resolution laser scanning microscope (Olympus, Japan).

Control and treated cells were lysed using RIPA lysis buffer (120 mM NaCl, 1.0% Triton X-100, 20 mM Tris–HCl, pH 7.5, 100% glycerol, 2 mM EDTA and protease inhibitor cocktail, Roche, Germany). Total protein (30 μg) was electrophoresed on 10% SDS–polyacrylamide gels and blotted onto PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% BSA at room temperature, the blots were probed with one of these antibodies: Par-4 (Cell signaling technologies, Santa Cruz Biotechnology, USA), PARP (Santa Cruz Biotechnology), GRP78 (Abcam, Cambridge, England), Akt, PKCζ, actin (Molecular probes, Invitrogen, USA), pAkt-ser 473, or pPKC ζ (Santa Cruz Biotechnology) overnight at 4 °C. The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and goat-anti-mouse IgG (Bio-Rad, USA). The immunoreactive bands were visualized by chemiluminescence using Super Signal West Femto Maximum Sensitivity Substrate (Pierce, USA). GAPDH and actin were used to normalize levels of proteins detected.
For secretion of Par-4, HNGC-2, LN-18 and G1 cells were exposed to tamoxifen for 24 h and supernatants were collected and concentration (20-fold) was achieved using 30 kDa cut-off filters (Millipore, USA).