Protein Extraction and Western Blot Analysis

Cells were collected by centrifugation, washed serially with cold water and lysis buffer (50 mM Tri-HCl, 500 mM NaCl, pH 7.4, supplemented with protease inhibitors: 1 mM PMSF, 4 mM benzamidine hydrochloride, 2.5 mM EDTA, pH 8) then disrupted with glass beads^{66 (link)}. Lysates were treated with 1% Triton X-100 on ice for 30 min and then with cracking buffer (8 M Urea, 5% (w/v) SDS, 40 mM Tris-HCl pH 6.8, 0.1 mM EDTA, 0.4 mg/ml bromophenol blue) at 37 °C for 10 min followed by incubation at 95 °C for a further 10 min. For western blotting, proteins were separated by electrophoresis on 10% (w/v) NuPAGE Bis-Tris gels (Life Technologies) before transfer to nitrocellulose membrane (GE Healthcare). Protein loading was shown by staining with Ponceau S (Sigma). Immunodetection of GFP tagged proteins was with anti-GFP primary antibody (1:1000 dilution; Roche) and poly horseradish peroxidase (poly HRP) conjugated goat anti-mouse antibody (1:10000 dilution; Thermo Scientific). GFP tagged proteins were detected with an electrochemiluminescence HRP kit (Pierce) and imaged using a Chemidoc XRS (Bio-Rad). Protein band intensities were quantified with ImageJ software.