Mouse anti c myc antibody

These were performed essentially as previously described (Long et al., 2016 (link)). HEK293 cells were co-transfected with the pRK5myc-hCB3_SH3-^R356Q construct at a 1:1 ratio with pEGFP-gephyrin using electroporation (Gene Pulser II, Bio-Rad). Cells were fixed after 24 h for 2 min in 4% (w/v) PFA in PBS. Immunostaining to detect collybistin was performed using a mouse anti-c-myc antibody (1:200, Sigma) and detected using an AlexaFluor 546 goat anti-mouse secondary antibody (1:600; Invitrogen). Counterstaining for cell nuclei was performed with DAPI (1:500; Life Technologies). Confocal microscopy was performed using a Zeiss LSM 710 META. All images were taken with a ×63 objective.

Human embryonic kidney (HEK293) cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum at 37°C, 5% CO₂ and transfected with 4 μg pRK5myc-hCB3_SH3− wild-type or R338W mutants using FuGENE (Roche). After 24 h, transfected cells were solubilized in a buffer containing Triton X-100 (Sigma-Aldrich), 1%; 150 mM NaCl; 50 mM Tris, pH 7.4, with protease inhibitor cocktail (Roche, Sussex, UK). Insoluble material was removed by centrifugation at 16, 100× g for 20 min. Phosphatidylinositol-3-phosphate (PI₃P/PtdIns-3-P) agarose beads (40 μl; Eschelon Biosciences) were incubated with cell lysates for 2 h at 4°C. Beads were washed four times in buffer. Proteins were eluted from beads by heating at 98°C for 3 min in 2 × sample loading buffer and then subjected to SDS-PAGE. Proteins binding to beads were detected by Western blotting using mouse anti-c-myc antibody (Sigma, 1:1000) and HRP-conjugated goat anti-mouse (Santa Cruz, 1:2000). Immunoreactivity was visualized using West Pico Chemiluminescent Substrate (Pierce). Expression levels of hCB3_SH3− and hCB3_SH3−R338W, and PI₃P pulldown assay results were assessed using an unpaired, two-tailed Student’s t-test.