Gfp pab

Cells seeded in flasks were trypsinized, washed with PBS, and lysed using Hunt’s buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5% NP-40) for 30 min on ice. For IP analysis, 1 mg lysate was continuously mixed with 2 μg antibody overnight at 4°C. Antibodies used were GFP pAb (Invitrogen, Thermo Fisher Scientific), APC Ab5 mAb (Merck Millipore, Darmstadt, Germany), APC C20 pAb (Santa Cruz), Miro1 pAb (Sigma-Aldrich, St. Louis, MO), immunoglobulin G (IgG) mouse (Sigma-Aldrich), and IgG rabbit (Sigma-Aldrich). Lysates were continuously mixed for an additional 2 h at 4°C with 30 μl protein A-Sepharose beads (Amersham Biosciences, GE Healthcare) to pull down immunocomplexes. Immunocomplexes attached to the beads were pelleted by centrifugation and washed three times with TBS. Precipitates were boiled in Laemmli buffer and subjected to SDS–PAGE and Western blotting as described below.

Antibodies and concentrations used for immunofluorescence microscopy (IF), Western blotting (WB), and Duolink PLA (D) are as follows. APC antibodies used were Ab1 mAb (1:100 WB; Merck, OP44), Ab7 mAb (1:300 D; Merck, OP80) and H-290 pAb (1:500 IF; Santa Cruz, sc7930). GFP antibodies used were GFP mAb (1:1000 IF and WB, 1:10000 D; Roche, 11814460001) and GFP pAb (1:1000 IF; Invitrogen, Thermo Fisher Scientific, ab27043). Other primary antibodies used were α-tubulin mAb (1:1500 IF and WB; Sigma-Aldrich, T9026), EB1 mAb (1:200 IF; BD, 610535), mtHSP70 mAb (1:500 IF and WB; Thermo Fisher Scientific, MA3-028), Miro-1 pAb (1:100 IF, 1:500 WB, 1:300 D; Sigma-Aldrich, HPA010687), Milton-2 pAb (1:100 IF, 1:500 WB, 1:300 D; Sigma-Aldrich, HPA015827), actin mAb (1:500 IF; Sigma-Aldrich, A2228), IQGAP1 mAb (1:500 IF; BD, 610612), and β-catenin mAb (1:100 IF; BD, 610154). If required, CMX-Ros (1:10000; Molecular Probes, Life Technologies) was added directly to seeded cells before fixation for 15 min at 37°C. Secondary antibodies used in immunofluorescence microscopy studies were Alexa Fluor 405 mAb (1:300; Invitrogen, Thermo Fisher Scientific), Alexa Fluor 488 mAb and pAb (1:500; Invitrogen, Thermo Fisher Scientific), and Alexa Fluor 594 mAb and pAb (1:2000; Invitrogen, Thermo Fisher Scientific).