Mouse anti α tubulin monoclonal dm1a

To generate whole cell lysates for Western blot analysis, cells were lysed in extraction buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 1 mM DTT, and 0.1 mM PMSF), extract protein concentrations were determined by Bradford, followed by addition of Laemmli sample buffer. Lysates were boiled for 5-10 min and stored at -20°C. Equal amounts of total protein of each whole cell lysate were resolved by SDS-PAGE, transferred onto nitrocellulose (Amersham), blocked in blocking buffer (5% milk in PBS, 0.1% Tween-20), sequentially probed with primary and secondary antibodies, and then scanned on a LiCor Odyssey CLx imager with Image Studio software (LiCor Biosciences). Primary antibodies used for Western blotting included rat anti-Cep135 (1:1000; McLamarrah et al., 2018) (link), guinea pig anti-Slimb (1:1000; Brownlee et al., 2011) (link), rat anti-Asl (1:1000; Boese et al., 2018) (link), rabbit anti-Ana2 (this study), mouse anti-V5 monoclonal (1:3000, Life Technologies), mouse anti-myc (1:3000, Cell Signaling), mouse anti-GFP monoclonal JL8 (1:3000, Clontech), and mouse anti-α-tubulin monoclonal DM1A (1:3000, Sigma-Aldrich). Primary antibodies were diluted as indicated in blocking buffer. Host-specific IRDye 800CW secondary antibodies (LiCor Biosciences) were prepared according to the manufacturer's instructions and used at 1:3000 dilution in blocking buffer.