Western Blot Analysis of Protein Expression

Cells were lysed in Laemmli buffer and analyzed for total protein concentration as described (Karni et al. 2007 (link)). Fifty micrograms of total protein from each cell lysate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% milk and probed with specific antibodies. Bands were visualized using enhanced chemiluminescence detection. Primary antibodies are as follows: hnRNP A1 (mAb A1/55, 1:1000) (Allemand et al. 2005 (link)), hnRNP A2/B1 (1:1000, Santa Cruz), β-tubulin (1:1000, Sigma), β-catenin (1:2000, Sigma), β-actin (1:1000, Santa Cruz), T7 tag (1:5000, Novagen), phospho-MEK S217/221 (1:1000, Cell Signaling), total MEK (1:1000, Cell Signaling), phospho-ERK T202/Y204 (1:1000 Sigma), total ERK1/2 (1:1000, Cell Signaling), A-Raf (1:1000, Cell Signaling), and A-Raf short (1:500) (Rauch et al. 2011 (link)). Secondary antibodies are as follows: HRP-conjugated goat anti-mouse, goat anti-rabbit, donkey anti-goat IgG (H+L; 1:10,000 Jackson Laboratories).