Anti myc agarose beads

Cells were washed with cold phosphate-buffered saline (PBS) and lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl and 0.5% NP-40) supplemented with protease inhibitors (K1019, APExBIO, Houston, TX, USA) and phosphatase inhibitors (K1015, APExBIO, Houston, USA). The cell lysates were clarified by centrifugation at 13,200 r.p.m. at 4 °C for 10 min. The protein concentrations of lysates were measured using Nanodrop with Bio-Rad protein assay reagent. Equal amounts of whole-cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 2000–5000 μg lysates were incubated with agarose-conjugated primary antibodies for 3–4 h at 4 °C or with the indicated primary antibody (3 to 5 μg) overnight followed by 1 h incubation with Protein A Sepharose beads. Immunoprecipitants were washed three times with NETN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and then resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-Flag agarose beads (A2220) were purchased from Sigma Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Anti-PRMT5 antibody conjugated to agarose (sc-376937) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cells were rinsed with ice-cold phosphate-buffered saline (PBS) and lysed in EBC buffer (50 mM Tris–HCl pH 7.5, 120 mM NaCl and 0.5% NP-40) or Triton buffer (40 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM MgCl₂, 1 mM EDTA and 1% Triton X-100) supplemented with protease inhibitor (Thermo Fisher, A32953) and phosphatase inhibitors (phosphatase inhibitor cocktail Set I and II, Calbiochem). The cell lysates were centrifuged at 13,200 r.p.m. at 4 °C for 10 min. The protein concentration of lysates was determined using Nanodrop by Bio-Rad protein assay reagent. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For IP, 2000–5000 μg lysates were incubated with agarose conjugated antibodies for 3–5 h at 4 °C. Immunoprecipitants were washed three times with NETN buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA and 0.5% NP-40) or Triton buffer before being resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-FLAG agarose beads (A2220) were purchased from Sigma-Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Some blots were cut prior to hybridization with primary antibodies, but one full-length original, unprocessed blot for each antibody was provided in the Supplementary Materials.