Hnrnp a2 b1

For Western analysis, proteins were resolved on Tris–Glycine gels, transferred to nitrocellulose or PVDF membranes (Millipore), and probed with the following antibodies: hnRNP A2/B1 (Novus NB120-6102), hnRNP K (Novus NBP2-24531), hnRNP A1 (Cell Signaling K350), hnRNP U (Abcam 180952), hnRNP L (Abcam 6106), β-actin (Thermo MA5-15739), EZH2 (Cell Signaling 3147), and α-tubulin (Santa Cruz sc-5286). Immobilon Chemiluminescent HRP Substrate from Millipore was used for detection using a BioRad ChemiDoc system. ImageJ was used to quantify each blot.

MDA-MB-231 or MCF-7 cells were washed in PBS and lysed in 10 mM Hepes 7.4, 150 mM KCl, 3 mM MgCl₂, 2 mM DTT, 0.5% NP-40, 1 mM PMSF, 10% glycerol with protease and RNase inhibitors (Roche). Immunoprecipitations were performed overnight at 4°C with 500–1000 µg of lysate and 5 µg of the following antibodies: Rabbit or Mouse Total IgG (Santa Cruz Biotechnology), hnRNP B1 (Immuno-Biological Laboratories Co. #18941), and hnRNP A2/B1 (Novus NB120-6102). For each IP, 25 uL of Protein A/G magnetic beads (Thermo-Fisher) were added to each sample and incubated an additional 2 h. Beads were washed 5× with the lysis buffer specified above, and split for RNA and protein analysis.