Anti acetylated tubulin clone 6 11b 1

Anti-GFP (Roche, 11 814 460 001), anti-HA (mouse hybridoma 12CAS), anti-Acetylated tubulin (Clone 6—11B-1, T6793, Sigma), anti-gamma tubulin (Clone GTU-88, Sigma), anti-A cyclase III (C-20, sc-588, Santa Cruz Biotechnology INC., Dallas, TX, USA), anti-Rab3IP (S-14, sn-162069, Santa Cruz Biotechnology Inc., Dallas, TX, USA).

Cultured MK were collected at day 3 and subjected to enrichment on a BSA gradient (1.5–3%). Cells (4 × 10⁶ cells) were washed in PBS, centrifuged at 100 g for 5 minutes and the pellets were resuspended in Laemmli buffer. Proteins from each sample were separated on 4–15% SDS gels (Bio-Rad, Hercules, CA), blotted onto PVDF membranes and incubated with specific primary antibodies. Specifically, anti-acetylated Tubulin Clone 6-11B-1 (Sigma Aldrich, Darmstadt, Germany), anti-α-Tubulin antibody clone DM1a (Sigma Aldrich, Darmstadt, Germany) and anti β-catenin clone 9581 (Cell signaling Technology, Danvers, MA) were used at 0.1 μg/mL, 1 µg/mL, and at a dilution of 1/1000, respectively. Membranes were then washed and incubated for 1.5 hour at room temperature with 50 ng/mL peroxidase-conjugated goat anti-rabbit or with peroxidase-conjugated goat anti-mouse IgG (Jackson Immuno Research, PA, USA) and 50 ng/mL peroxidase conjugated goat anti GAPDH (Abcam, Paris, France) at room temperature and resolved by Clarity Western ECL Substrate (Bio-Rad, Hercules, CA). Membranes were visualized on a Chemidoc Imaging System (Bio-Rad, Hercules, CA).