Ab109451

For immunoblot analysis: Antibodies purchased from Cell Signaling include: PRMT5 Ab (2252 s, 1:1000), Total Rb (4H1) mAb (9309 s, 1:1000), pRB/s807/811 (D20B12) mAb (8516 s, 1:1000), β-actin (13E5) mAb (4970 s, 1:5000), Vinculin (E1E9V) mAb (13901 s, 1:2000), SDMA Ab (13222 s, 1:1000), MEP50 Ab (2823 s, 1:1000), PRMT5 (D5P2T) mAb (79998 s, 1:1000), and ORC2 (3G6) mAb (4736 S, 1:1000); Antibodies purchased from Santa Cruz Biotechnology include: ERα (F-10) mAb (sc-8002, 1:1000) and POLE (D-10) mAb (sc-390785, 1:1000); Antibodies purchased from Abcam: PRMT5 (EPR5772) mAb (ab109451, 1:1000) and GINS1 (EPR13359) mAb (ab181112, 1:1000); FUS Ab was purchased from Proteintech (11570-1-AP, 1:1000); Total Pol II (4H8) mAb was purchased from MilliporeSigma (05-623, 1:1000); APC7 Ab was purchased from Bethyl Laboratories (A302-551A, 1:1000). For Co-IP: PRMT5 mAB (EPR5772) was purchased from Abcam (ab109451, 1:250); FUS mAB (4H11) was purchased from Santa Cruz Biotechnology (sc-47711, 1:100). For ChIP-seq: pSer2 Pol II Ab was purchased from Abcam (ab5095, 1:40). For IHC, SDMA Ab was purchased from Cell Signaling (13222 s, 1:600); ERα mAb (F-10) was purchased from Santa Cruz Biotechnology (sc-8002, 1:800); Ki67 (MIB-1) mAb was purchased from Agilent (IR62661-2, ready to use).

Whole-cell lysates of HCT116 and SW480 cells were extracted using lysis buffer, including 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were centrifuged at a speed of 14000 rpm for 15 min, then mixed with 5×loading buffer and denatured at 100 °C for 5 min. Aliquots of supernatant protein (40 ~ 50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The PVDF membranes were then blocked for 1 h with 5% (w/v) non-fat milk at room temperature. After incubation with anti-PRMT5 (Abcam, ab109451), anti-EZH2 (ProteinTech, 21800-1-AP), anti-CDKN2B (Abcam, ab53034) or anti-GAPDH (ProteinTech, 60004) overnight at 4°C, the PVDF membranes were washed with PBST and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich) at room temperature. Subsequently, the immunoreactive bands were visualized using ECL detection reagent (Thermo Scientific).