Cyclin d3

Cells were lysed in RIPA buffer [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA] including protease inhibitor cocktail (Millipore, #539137) on ice followed by short sonication. Sample supernatants were collected after centrifugation at 14,000 rpm for 10 min. Forty micrograms of protein were separated on a 10% SDS-polyacrylamide gel before being transferred to nitrocellulose using a Bio-Rad Mini-Blot transfer apparatus (Richmond, CA). Membranes were blocked in Tris-buffered saline (TBS) containing 5% non-fat milk powder (Lab Scientific, Livingston, NJ) for 1 h.
Immunoblotting was performed at 4 °C overnight using antibodies against ACSL3 (ab151959), ACSL4 (ab205197), CD34 (ab81289) from Abcam (Cambridge, MA), AR (N-20) from Santa Cruz Biotechnology (Dallas, TX), CDK4 (Cat# 12790T), cyclin D1 (Cat# 2978), cyclin D3 (Cat# 2936T), GAPDH (Cat# 2118) from Cell Signaling (Boston, MA), or NMT1 (HPA022949) from Sigma (St. Louis, MO). The membranes were incubated with a secondary antibody at room temperature for 1 h after washing with TBS containing 0.5% Tween-20. Protein bands were visualized using the Pierce ECL Western Blotting substrate (Rockford, IL) and quantified by Image J software.

Cells were treated with DMSO, TA, VCR or combination for 24 and 48 h. Total cellular protein extracts and western blotting was then done using the previously described method (Abdelrahim et al., 2006 (link)). Proteins of interest were probed by specific primary antibodies of apoptotic markers, cleaved poly-ADP-ribose polymerase (c-PARP, Cell Signaling Technology, Danvers, MA) and survivin (R&D Systems, Minneapolis, MN), and cell cycle markers cyclin A, cyclin D3 (Cell Signaling Technology), cyclin B1 and cyclin dependent kinases 4/6 (CDK4/6, Santa Cruz Biotechnology, Santa Cruz, CA). The expression of β-actin (Sigma) was used as a loading control.