Protein expression and co-immunoprecipitation in HEK293T cells

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, 100 mg/L streptomycin and 100 IU penicillin, in humidified 5% (v/v) CO₂ air at 37 °C. Cells were seeded at a density of approximately 8 × 10⁵ cells/6-cm plate and the transfection were carried out with a calcium phosphate precipitation protocol. Briefly, different combinations of plasmid DNA (2~5 μg / construct) were mixed with 30 µl 2.5 M CaCl₂ and ddH₂O to a total volume of 300 µl, then 300 µl of 2x HeBS [250 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, 12 mM Dextrose and 50 mM HEPES, adjust the pH of the final solution to 7.05] was added drop by drop with vortex, and kept at room temperature for 5 min before applying to cells. The media were aspirated from each plate, DNA mixtures were gently added into plates and the plates were rotated gently to allow the mixtures to coat the entire plate. 3 ml of fresh media containing 25 μM chloriquine (cat # C6628, Sigma) were added to each plate and the plates were kept in the CO₂ incubator overnight. The next day, the media were changed with 3 ml of fresh media without chloriquine. 36–48 h after transfection, the cells were subjected to blue light treatment for indicated time, washed twice with cold PBS buffer and then harvested in liquid nitrogen for co-immunoprecipitation.

Cells transfected with different plasmid DNA were lysed in 800 μl 1% Brij buffer [1% Brij-35, 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA,1x Protease inhibitor cocktail, and 1x phosphatase inhibitor PhosSTOP] with rotating at 4 °C for 20 min. Cell lysates were centrifuged at 16,000 rcf for 10 min at 4 °C, and the supernatants were incubated with 20 μl Anti-FLAG M2 affinity gel (Cat. # F2426, Sigma) at 4 °C for 2 h with rotation. Beads were washed with 1% Brij buffer for 5 times. Proteins were competed from the beads with 35 μl 3xFlag peptide solution [500 ng/μl in 1% Brij buffer] for 30 min at room temperature with mixing. Elution was transferred to a new tube, mixed with 4XSDS sample buffer, denatured at 100 °C for 4 min and subjected to western blot analysis. Western blots were performed as described above. Primary antibodies used here were anti-Flag (1:1500, cat # F1804, Sigma), anti-Myc (1:5000, cat # 05-724, Millipore), and anti-HA (1:5000, cat # 12013819001, Roche), and secondary antibodies used were anti-Mouse-HRP (1:10000, cat # 31430, ThermoFisher) and anti-Rabbit-HRP (1:10000, cat # 31460, ThermoFisher).