Co-immunoprecipitation assays was carried out with HEK293 cells homogenized in 0.4 mL of lysis buffer and centrifuged for 30 minutes at 4°C and 16,100 xg. Supernatants were transferred to fresh centrifuge tubes, and 10 μL Protein A-Sepharose beads (GE17–0780-01, Millipore, MA) and 1.0 μg of control IgG antibody (sc-66931, SCBT, CA) were added. Tubes were incubated for 1 hour at 4C to preclear samples. Precleared samples (1 mL) were incubated with 30 μL Protein A-Sepharose beads and 2 μg of anti-Flag M1 (Sigma-Aldrich, # F3040) or anti-IgG antibody (sc-66931, SCBT, CA) at 4°C overnight. After incubation, beads were rinsed with lysis buffer 3 times. The washed beads were mixed with 30 μL 2x SDS loading buffer (#161–0747, Bio-Rad Laboratories, CA), subjected to electrophoresis, and Western blotted for FLAG-β2AR, Nb80, and PDE4D5. Gel images were taken and quantified using a Bio-Rad ChemiDoc MP Imager.
Martinez J.M., Shen A., Xu B., Jovanovic A., de Chabot J., Zhang J, & Xiang Y.K. (2023). Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory. Science signaling, 16(778), eade3380.
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