Anti adma

Cells and tumor tissues were collected and lysed as described before^{44 (link)}. Briefly, 20–100 μg of protein was boiled at 95 °C for 5 min, loaded onto NuPAGE 4–12% Bis-Tris protein gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad) using the Mini-PROTEAN Tetra electrophoresis system (Bio-Rad). Primary antibodies used for membrane staining were anti-PRMT1 (Millipore, 7404; 1:1,000), anti-ADMA (Cell Signaling, 13522S; 1:1,000), anti-GAPDH (Santa Cruz Biotechnology, sc-32233; 1:5,000), anti-pH2A.X (Cell Signaling, 9718S; 1:1,000), anti-β-actin (Santa Cruz Biotechnology, sc-47778; 1:3,000) and anti-dsRNA (J2) (Scicons, 10010200; 1:5,000). Images were collected on an Odyssey scanner (LiCor) or Amershan ImageQuant 80 and analyzed with ImageQuant TL (v8.2.0) and ImageJ (v1.53a).

The human VMRC-LCD cells, LCD cells stably expressing GFP/GFP-TDRD3 and MDA-MB-231 cells expressing Tet-on-sh TDRD3 have been described before [13 (link), 14 (link)]. Wild-type and Tdrd3-knockout MEF cells were generated from E12.5 mouse embryos following a standard MEF-generation protocol and the primary MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. The HeLa, HEK293 and MDA-MB-231 cell lines were obtained from ATCC. All of the cell lines were maintained in DMEM containing 10% fetal bovine serum. The anti-TDRD3 antibody has been described before [13 (link)]. The anti-TDRD3 (CST) (cat#5942), anti-USP9X (cat#14898), anti-ADMA (cat#13522) and anti-SDMA (cat#13222) antibodies were purchased from Cell Signaling Technology. Anti-MCL-1 (cat# sc-819) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-ACTIN (cat# A5316) was from Sigma Aldrich (St. Louis, MO, USA). Anti-PRMT1 (cat# A300722A) was from Bethyl Laboratories (Montgomery, TX, USA). Anti-TIAR (cat# 610352) and anti-G3BP (cat# 61126) were from BD Biosciences (San Jose, CA, USA).