H3r2me2a

Equal amounts of colon tissue or cancer cell extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with antibodies against PRMT6 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), cleaved PARP (1:1000; Cell Signaling Technology), β-actin (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mono- and dimethyl arginine (1:500; Abcam, Cambridge, UK), p21 (1:1000, Cell Signaling Technology), p53(1:5000, Santa Cruz), histone H3 (1:50000, Abcam), H3R2me2a (1:500, Novus Biologicals, Littleton, CO, USA). Subsequently, the membranes were incubated with the secondary antibodies for 1 h at room temperature, and an Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for detection. The relative protein levels were calculated by comparison to the levels of β-actin.

Protein was extracted from whole-cell lysates using Kinexus Lysis Buffer with Lysis Buffer Cocktail (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada) and protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Subsequently, 30 μg of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with antibodies against PRMT6, H3R2me2a (1/500 Novus Biologicals, Littleton, CO), H3K4me3, PSA (1/1500 and 1/6000 Abcam, Cambridge, UK), AR, mTOR (1/1000, Cell Signaling Technology, Inc., Danvers, MA), p21, p27 (1/500, BD Biosciences, Franklin Lakes, NJ), AKT (1/500, Santa Cruz Biotechnologies Inc) and pAKT (1/500, Millipore Billerica, MA), as well as histone H3 (dilution: 1:500, Abcam) and beta-actin (dilution: 1:8000, Sigma-Aldrich) as input controls, as appropriate. Blots were developed using Immun-Star™ WesternC™ Kit (BioRad, Hercules, CA). All experiments were performed in triplicate.