Anti prmt5

Cells were washed with cold PBS, and then lysed in RIPA buffer (Cell Signaling Technology). After centrifugation at 14,000 rpm for 15 minutes at 4°C, the supernatants were collected, and the protein concentrations were measured using BCA protein assay reagent (BIO-RAD). Subsequently, equal amounts of proteins were separated in NuPAGE 4-12% Bis-Tris gradient gel (Invitrogen #NP0335), and transferred onto nitrocellulose membranes (Invitrogen #B301002). After blocking with 5% milk, the membranes were then probed at 4°C overnight with various primary antibodies: anti-γ-H2AX (Cell Signaling), anti-phospho-Chk1 (Ser345) (Cell Signaling), anti-phospho-Chk2 (Thr68) (Cell Signaling), anti-PRMT5 (Abcam), anti-RPA1 (Abcam), anti-phospho-RPA2 (S4/S8) (Bethyl Laboratories), anti-RPA2 (Ab-2) (Calbiochem), cleaved caspase-3 (Cell Signaling), and anti-β-actin (Sigma), washed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween 20; pH 7.6), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega) at room temperature for 1 hour. Finally, after washing with TBST, the antibody-bound membranes were treated with enhanced chemiluminescent western blot detection reagents (GE Healthcare) and visualized with an x-ray film (GE Healthcare).

Cells were seeded at a density of 5 × 105 cells/ml; at around 60% confluency, cells were treated and treated as indicated above. Cells were washed twice with PBS and Cells stained with anti-PRMT-5 (Abcam, UK), overnight at 4 °C. Cells were then washed with 1X PBS and reacted with the Alexafluor®488-labeled secondary antibody (Abcam, Cambridge, United Kingdom) for 1 h at 37 °C; excess reagent was rinsed with 1X PBS. Genomic DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, California, United States) according to manufacturer's instructions. Slides were visualized by confocal microscopy using a Nikon Confocal Microscope (Nikon, Tokyo, Japan).

Cells were lysed in ice-cold NP-40 lysis buffer (1.0% NP-40, 150 mM of NaCl, 50 mM of Tris-Cl, pH 8.0) containing protease inhibitor cocktail tablets (Cat. No. S8830; Sigma, Germany). Protein concentration of cell lysate was quantified using the standard Bradford method (Cat. No. 500-0006; Bio-Rad, Hercules, CA, USA). 50 µg of lysate protein aliquots were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, USA). 5% skimmed milk powder was used to block the membrane at room temperature for 1 h. The membrane was then washed with TBST, and incubated with the following primary antibodies (anti-PRMT-5, anti-β-catenin, anti-H4R3me2s [Symmetrical dimethylation on arginine-3 of histone H4], anti-β-actin (all from Abcam, UK), ( cyclin D1, cdk4, cdk6, caspase-3 and PARP, all from Cell Signaling Technology, USA) overnight at 4 °C. Secondary antibodies (Cell Signaling Technology, USA) were incubated with the membrane at 1:1000 dilution for 1 h at room temperature. Chemiluminescence was detected using the ECL kit (Thermo Scientific Pierce, USA). Bio-Rad Image Lab software (ChemiDoc™ Touch Gel and Western Blot Imaging System; Bio-Rad) was used to detect and quantify protein bands. Protein levels were normalized to β-actin and ratios were calculated based on the values of control (untreated) samples.