Hnrnpc1 c2

The following abs were used at the indicated final concentration or dilution: mouse monoclonal abs to hnRNP U (Santa Cruz; 3G6; 0.2 μg/ml), hnRNP A1 (Sigma; 4B10; 2 μg/ml), lamin B1 (Calbiochem; 101-B7; 1/1,000), α-tubulin (Sigma; B-5-1-2; 1/1,000), G3BP1 (BD; 23/G3BP; 0.25 μg/ml), hnRNP C1/C2 (Sigma; 4F4; 0.4 μg/ml), β-actin (Developmental Studies Hybridoma Bank; 1/1,000), HuR (Santa Cruz; 3A2; 0.2 μg/ml); rabbit antisera to HMGB2 (Abcam; 1/1,000), ICAD (Abcam; 0.5 μg/ml), NE (Abcam; 1 μg/ml); DDX5 (Abcam; 0.5 μg/ml). Secondary abs were sheep anti-mouse-HRP (GE; 1/2,500), donkey anti-rabbit-HRP (GE; 1/2,500), donkey anti-goat-HRP (GE, 1/2,500), goat anti-rabbit AF488 (Invitrogen; 1/200), donkey anti-mouse Cy3 (Jackson ImmunoResearch 715-165-150; 1:200). Normal rabbit IgG (Cell Signaling 2729; 1 μg/ml) was used as an isotype control for immunofluorescence.

Cells were resuspended in RIPA buffer: 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM dithiothreitol, 0.5 mM NaVO4, and protease inhibitor cocktail (Sigma-Aldrich). After 10 min on ice, extracts were centrifuged for 10 min at 12,000 g ,supernatants were collected and used for western blot as described (49 (link)). Primary antibody incubation (1:1000) was carried out with the following antibodies: PKM1, hnRNPA1, hnRNPA2/B1, hnRNPC1/C2 (Sigma-Aldrich); PKM2 (Cell Signaling Technology, MA, USA); PTBP1, SRSF1, SRP20, SRp40/p55/p75 (Santa Cruz Biotechnology, CA, USA); hnRNPF/H (Abcam, UK). PTBP2 antibody was a generous gift of Professor Douglas L. Black (UCLA, CA). Images of the western blot were acquired as TIFF files.