Rabbit anti numa

Cells in 6-well plates were lysed, and protein extracts were collected after centrifugation at 4°C for 30 min. Protein concentrations were measured using a Bradford assay, and equal concentrations of each sample were separated on a 4–12% Bis-Tris gel (Invitrogen) by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 4% milk, incubated in primary antibodies overnight at 4°C, and incubated with HRP-conjugated secondary antibodies for 1 hour. Proteins were detected using SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Fisher). The following primary antibodies were used: mouse anti-α-tubulin DM1α (1:5,000, Sigma-Aldrich T6199), rabbit anti-cyclin D1 SP4 (1:1,000, Abcam ab16663), rabbit anti-cyclin D1 E3P5S (1:1,000, Cell Signaling 55506), rabbit anti-TACC3 D9E4 (1:1,000, Cell Signaling 8069), rabbit anti-Aurora A kinase (1:1,000, Cell Signaling 3092), rabbit anti-NuMA (1:1,000, Novus NB500-174), mouse anti-KIFC1 M-63 (1:1,000, Santa Cruz Biotechnology sc-100947), and rabbit anti-Vinculin E1E9V (1:10,000, Cell Signaling 13901). The following secondary antibodies were used at a 1:10,000 dilution: goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2005) and mouse anti-rabbit IgG-HRP (Santa Cruz Biotechnology sc-2357).