Immunoblot samples were pelleted and lysed for 30 min at 4°C in 1% (vol/vol) Triton X-100 (Sigma-Aldrich) and 1 mM MgCl2 in PBS (Gibco) supplemented with 1× cOmplete protease inhibitors (Roche) and 0.2% (vol/vol) Benzonase (Merck). An equal volume of 2X reducing SDS sample buffer was added, and 15 μl samples were resolved on NuPAGE 4–12% Bis–Tris gels (Invitrogen) in 1X MOPS (Thermo Fisher Scientific). Proteins were transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked in 5% (wt/vol) milk powder in Tris-buffered saline containing 0.1% (vol/vol) Tween-20 (TBS-T) for 1 h, then incubated with primary antibodies diluted in 5% milk/TBS-T overnight at 4°C. Primary antibodies used were as follows: rabbit α-HA (3F10, 1:1,000, Cat# 11867423001, RRID:AB_390918; Roche); mouse α-FLAG (1:1,000, Cat# F1804, RRID:AB_262044; Sigma-Aldrich); and rabbit α-GAP45 (1:1,000) (Gaskins et al, 2004 (link)) (Table S7). Membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies (Southern Biotech; see Table S8) diluted in 5% milk/TBS-T at 1:1,000 for 1 h before washing with TBS-T. Clarity Western ECL Substrate (Bio-Rad) was applied directly to membranes, and proteins were visualised using the ChemiDoc Gel Imaging System (Bio-Rad).
Table S7.
Table S8.
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