Super signal west dura ecl detection reagent

Western blot was performed as described previously^{69 (link)} with minor modifications. Briefly, protein samples were heated at 95 °C for 5 min with 2xSDS loading buffer (Invitrogen) and resolved on 12% SDS-PAGE (Invitrogen). Following electrophoresis, the proteins were transferred to PVDF membrane 0.2 µm (Millipore). Primary mouse anti-GFP antibodies (dilution WB 1:1000) (#11814460001, Roche) and secondary goat anti-mouse IgG (H + L) conjugated with HRP (dilution WB 1:5000) (Cat. No. G-21040, Life Technologies) were used for immunoblotting. Bound primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and the Super Signal West Dura ECL detection reagent (Life Technologies). The specific protein bands were visualized using the Super Signal West Dura ECL detection reagent (Life Technologies) and imaged using the ChemiDoc Imaging System (Bio-rad, Gladesville, New South Wales, Australia).

For SDS-PAGE, cells were harvested, rinsed in PBS and were lysed in lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% Triton X-100 with protease and phosphatase inhibitors. Lysates were collected by scraping and cleared by centrifugation at 4 °C. The protein content of all extracts was determined using a BCA protein assay kit (Thermo Fisher Scientific, Victoria, Australia) using bovine serum albumin (BSA) as the standard. Thirty micrograms of cellular protein were resolved by 10% SDS-PAGE and were transferred to PVDF membranes (Millipore). Bound IgG was visualized with horseradish peroxidase-conjugated secondary antibodies and the Super Signal West Dura ECL detection reagent (Life Technologies) and was imaged using the ChemiDoc Imaging System (Bio–rad, Gladesville, New South Wales, Australia). Uncropped and unprocessed scans of the all blots presented in the main figures are available as a Supplementary Fig. 11 in the Supplementary Information.