Protease inhibitor mix hp plus

HeLa Kyoto and derivatives were lysed into a buffer containing 20mM Tris-HCl, pH 8.0, 100mM KCl, 1% Triton X-100 (Splinter et al., 2012 (link)), benzonase (25 U/mL) and a protease-inhibitors cocktail (Protease-inhibitor mix HP Plus, Serva). Lysates were diluted at 0.5 mg/ml into Laemmli buffer, boiled at 95°C and resolved on a 6% SDS-PAGE before transfer onto a nitrocellulose membrane. After blocking with 5% milk in PBS-Tween (0.1%), membranes were probed with antibodies diluted as follows: anti-alpha-tubulin DM1α, 1:5000 (Sigma-Aldrich); rabbit polyclonal anti-GFP, 1: 5000 (Abcam); mouse anti-p150^Glued, 1:1000 (BD Transduction Laboratories); Sheep anti-mouse HRP and Donkey anti-rabbit HRP, 1:5000 (Amersham). All membranes were incubated with ECL reagent and developed onto Hyperfilm (Amersham) or imaged with the ChemiDoc™ MP Imaging System (BIO-RAD). The levels of images from scanned Hyperfilm and ChemiDoc tiff-converted files were adjusted using FIJI and converted to 8-bit. Western blots against the dynein HC were performed as above with the following modifications; lysates were diluted to 0.75 - 1mg/ml in Laemmli buffer, blocking buffer was 5% BSA in PBS-Tween (0.1%), membranes were probed with Rat dynein HC antibody (Santa Cruz Biotechnology) at 1:250 dilution.

HeLa cells were grown in DMEM supplemented with 10% tetracycline-free fetal bovine serum, 1% L-glutamine and 1% Penicillin-Streptomycin at 37°C in a 5% CO₂ atmosphere. Cells were treated with 50 ng/mL doxycycline (Sigma) for 18 hr and then treated with both 50 ng/mL doxycycline and 9 µM CDK1 inhibitor RO-3306 (Merck) for additional 6 hr before harvest. Cells were washed twice with PBS, resuspended in lysis buffer containing 75 mM HEPES pH 7.5, 1.5 mM EGTA, 1.5 mM MgCl₂, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF and Protease Inhibitor Mix HP Plus (SERVA), lysed using Bioruptor Plus sonication device (Diagenode) and centrifuged at 16,000 x g for 30 min at 4°C. The supernatant containing 2 mg protein was incubated with 7.5 µl GFP-Trap_A beads (Chromotek) for 2 hr at 4°C. The beads were washed thrice with the lysis buffer containing 300 mM NaCl instead of 150 mM NaCl. Proteins were eluted by adding SDS sample buffer and were analyzed using Tricine-SDS-PAGE and Western blot analysis. EGFP-M18BP1, mCherry-M18BP1, mCherry-Mis18α, Mis18β and vinculin were detected using following antibodies: anti-GFP (Abcam, AB6556), anti-mCherry (Novus, NBP1-96752), anti-Mis18β (Atlas, HPA052271), anti-vinculin (Sigma, V9131) anti-mouse-HRP (Amersham, NXA931-1ML) and anti-rabbit-HRP (Amersham, NXA934-1ML).