Ha antibodies

HEK293T cells were co-transfected with Flag-tagged target protein, HA-tagged source protein, and GFP-tagged peptide or GFP. Cells were lysed 48 h after transfections with radioimmune precipitation assay buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 10 mM Na₃VO₄, 10 mM sodium pyrophosphate, 25 mM NaF, 1× protease inhibitor mixture (Sigma)) for 30 min at 4 °C and co-immunoprecipitated with Flag beads (Clontech). The resulting immunocomplexes were analyzed by immunoblot using the appropriate antibodies. Protein samples were separated using 4–20% Mini-PROTEAN Tris-glycine gels (Bio-Rad) transferred to PVDF membranes and blocked in 5% milk containing PBS-Tween-20 (0.1%) for 1 h. PVDF membranes were then incubated with specified primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse HRP-linked, Cell Signaling, catalog #7076, 1:5000; anti-rabbit HRP-linked, Cell Signaling, catalog #7074, 1:5000) and detected using enhanced chemiluminescence (GE Healthcare). HA antibodies were obtained from Santa Cruz (catalog #7392, 1:2000), GFP antibodies were purchased from Abcam (catalog #ab290, 1:2000), and Flag antibodies were purchased from Sigma (catalog #A8592, 1:1000).