Mab10764

Primary antibodies used were mouse anti-SBP (1:1,000; MAB10764; EMD Millipore), rabbit antiactin (1:3,000; 8227; Abcam), mouse anti-GFP (IB, 1:1,000; immunofluorescence [IF], 1:2,000; 11814460001; Roche) mouse anti-V5 (1:2,000; R960-25; Invitrogen), rabbit anti-GM130 (1:500; ab30637; Abcam), goat anti–GMAP CRB16 and goat anti–Golgin245 CRB14 (1:2,000; Riedel et al., 2016 (link)), rabbit anti-Sec16 (1:2,000; Ivan et al., 2008 (link)), rabbit anti-TRAPPC9 AC1, rabbit anti-TRAPPC12 AC1 (IB, 1:1,000), rabbit anti-TRAPPC12 AC2 (IF, 1:100), and rabbit anti–Drosophila AP-1–γ (Hirst et al., 2009 (link)). Secondary antibodies were species-specific donkey anti-IgG, goat anti-IgG1, and goat anti-IgG2a antibodies coupled with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:100; Thermo Fisher Scientific) and anti-IgG antibodies coupled with horseradish peroxidase (Santa Cruz Biotechnology, Inc.). GFP was also detected with the GFP booster Atto488 (1:400; gba488-100; ChromoTek).

Tandem affinity purification was performed using the InterPlay mammalian TAP system (Agilent Technologies) according to the manufacturer's instructions, with some modifications. Briefly, TRex-TAP-eEF1A1 and TRex-TAP-eEF1A2 cells were induced with Dox, harvested, lysed and the lysates, containing TAP-tagged proteins, were subjected to affinity-based chromatography using streptavidin-linked resin only. Proteins eluted from streptavidin-resin were stored in the presence of 1 mM PMSF (Sigma-Aldrich) and 1× Complete (ethylenediaminetetraacetic acid (EDTA)-free) protease inhibitor cocktail (Roche) to promote protein stability. Protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The presence of streptavidin binding protein (SBP)-tag was checked by western blot and probing with a mouse monoclonal anti-SBP-tag antibody (Merck-Millipore, MAB10764).