Prmt1

PAK4 (Cell Signaling Technology, USA); PAK4 (Santa cruz); Phospho-PAK4 (Ser474,Cell Signaling Technology, USA); RUNX1 (Abcam, USA), RUNX1 (Santa cruz), SIN3A (Cell Signaling Technology, USA); HDAC1 (Cell Signaling Technology, USA); PRMT1 (Cell Signaling Technology, USA); Mono-Methyl Arginine (R*GG) (Cell Signaling Technology, USA); Jagged-1 (Cell Signaling Technology, USA); PTHrP (Abcam, USA); Flag, GFP, GAPDH (GenScript, Nanjing, China).

Total proteins were extracted from cells and tissue using RIPA Lysis Buffer (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 1% PMSF (Sigma). The concentration of the protein samples was determined and standardized using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Thirty micrograms of each lysate was electrophoresed by SDS-PAGE on 10% SDS gels, and then were transferred onto a PVDF membrane. After over 1 h of blocking in 5% milk with TBST buffer (20 mM Tris-HCL, 137 mM NaCl, 0.5% Tween 20, pH 7.6), the membrane was incubated with the appropriate primary antibodies of P-gp (Cell Signaling Technology Inc, Beverly, MA, USA), PXR (Abcam, Cambridge, MA, USA) and PRMT1 (Cell Signaling Technology Inc, Beverly, MA, USA) at 4°C overnight. Then, the membrane was washed with TBST buffer for 3 times (10 min each time), and incubated with the corresponding peroxidase-conjugated secondary antibodies (1:2000) for 1 h at 37°C. The membrane was washed again, 3 times with TBST and twice with TBS (10 min each time), and then was exposed to Immobilon Western Chemilum Hrp Substrate (Millipore, Boston, MA, USA). Finally, protein concentration was estimated relative to β-actin (for PXR and P-gp) or tubulin (for PRMT1) using WCIF Image J Software [24 (link)].