For pErk, E-Cadherin, Vimentin, and tubulin blots, cells were lysed in 50 mM Tris pH 7.5, 10 mM MgCl2, 0.5 M NaCl, and 2% Igepal (Cytoskeleton, Denver, CO) containing protease inhibitors and phosphatase inhibitors. Lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies were used according to manufacturer recommendations: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) (Cell Signaling Technology), E-Cadherin (4A2) (Cell Signaling Technology), Vimentin (D21H3) (Cell Signaling Technology), and α-Tubulin (clone B-5-1-2) (Sigma). Secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used according to manufacturer recommendations.
For GALNT7 blots, cells were lysed in Laemmli buffer. Great than 50 μg of lysate was subjected to SDS-PAGE and transferred to nitrocellulose membrane. Anti-GALNT7 antibody (ab97645) (Abcam) was used at 1:200 in PBS containing 0.1% tween after blocking in 5% milk, overnight at 4 °C. Anti-Rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE) was used according to manufacturer recommendations.
For GALNT7 blots, cells were lysed in Laemmli buffer. Great than 50 μg of lysate was subjected to SDS-PAGE and transferred to nitrocellulose membrane. Anti-GALNT7 antibody (ab97645) (Abcam) was used at 1:200 in PBS containing 0.1% tween after blocking in 5% milk, overnight at 4 °C. Anti-Rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE) was used according to manufacturer recommendations.