Endogenous BiP-3xFLAG IP

BiP-3xFLAG HEK293 cells (0.9 million cells/well) were plated on poly-lysine coated 6-well plates and transiently transfected with 2 μg of indicated plasmids and induced with doxycycline (200 ng/ml) for 24 h. The cells were starved with Met/Cys-free medium, including 2.5% dialyzed FBS and doxycycline for 45 min. Cells were then labeled with 90 μCi/ml Express-³⁵S protein labeling Mix (NEG772007MC) for 2 min at 37°C. The labeled cells were immediately placed on ice and harvested in 1 ml of ice-cold 1XPBS and centrifuged at 10,000 g for 2 min. Cell pellets were solubilized in 200 μl of lysis buffer (50 mM Tris, pH 7.5, 160 mM NaCl, 1% Digitonin, 1 mM CaCl₂, Apyrase (10 U/ml)) for 30 min on ice. The lysate was diluted to 1 ml with lysis buffer containing 0.1% digitonin before centrifugation at 20,000 g for 15 min. While 800 μl of supernatant was incubated with 12 μl of mouse anti-FLAG beads (Sigma-Aldrich), the remaining 200 μl was diluted to 800 ul with NP40 buffer and incubated 15 μl mouse anti-HA magnetic beads (Thermo Fisher Scientific). After 90 min of incubation, anti-FLAG beads were washed 3× with 1 ml of lysis buffer containing 0.1% digitonin, but anti-HA magnetic beads were washed with 3× with 1 ml of NP40 buffer. The washed beads were boiled with 50 μl of 2× SDS samples buffer and analyzed by 7.5% Tris-Tricine SDS-PAGE and autoradiography. For the second IP, anti-FLAG immunoprecipitants (∼40 μl in 2× SDS sample buffer) were diluted to 1 ml with Triton buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100) and incubated with 15 μl of anti-HA magnetic beads for 90 min. The beads were washed three times with 1 ml of Triton buffer and analyzed as above.