Anti ha tag antibody conjugated agarose beads

Coimmunoprecipitation assays were performed essentially as described previously (Kobayashi and Fukuda, 2013a (link)). In brief, lysates of COS-7 cells expressing HA–Rab35-Q67L were incubated for 1 hr at 4°C with anti-HA tag antibody-conjugated agarose beads (Sigma–Aldrich Corp.) (wet volume 10 µl). After washing the beads with 1 ml of washing buffer, the beads coupled with HA–Rab35-Q67L were incubated for 1 hr at 4°C with cell lysate expressing FLAG–Rab1A-Q70L, FLAG–Rab8A-Q67L, FLAG–Rab8B-Q67L, FLAG–Rab13-Q67L, or FLAG–Rab36-Q116L, in the absence or presence of cell lysate expressing T7–MICAL-L1. After washing the beads with the washing buffer, the FLAG–Rab1A-Q70L, FLAG–Rab8A-Q67L, FLAG–Rab8B-Q67L, FLAG–Rab13-Q67L, FLAG–Rab36-Q116L, and T7–MICAL-L1 bound to the beads were analyzed by SDS–PAGE followed by immunoblotting with HRP-conjugated anti-FLAG tag antibody and HRP-conjugated anti-T7 tag antibody. The immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare Ltd). Input means 1/500 volume of the reaction mixture used for coimmunoprecipitation. The blots shown in this paper are representative of at least three independent experiments.

For transient transfection, 293T cells were co-transfected with HA-FOXR2/SRF and Myc-EPC1/EPC2 expression vectors. At 36 to 48 h post transfection, cells were collected for immunoprecipitation assays. The primary antibodies anti-Myc-Tag, anti-HA-Tag and anti-β-Actin, and secondary antibodies HRP-labeled goat anti-rabbit IgG (H + L) (Cat# A0208, Beyotime) and HRP-labeled goat anti-rat IgG (H + L) (Cat# A0192, Beyotime) were used. Anti-HA-Tag antibody-conjugated agarose beads were purchased from Sigma (Cat# A2095). After addition of cell extracts (transfected with Myc tagged EPC1/EPC2 and HA tagged FOXR2/SRF) and incubation for 4 h at 4°C, the bound beads were washed with PBS and analyzed by WB analysis. The Amersham Imager 600 analyzer was used for photographing the blots. ImageJ software was used for quantifying the protein levels based on the band density obtained in the WB analysis.