Flag-CRTC3 was expressed in HEK293 cells. CRTC3 was immunoprecipitated from 0.25 mg cell extract protein with anti-FLAG or different antibodies against CRTC3 (2.5 ug). Immunoprecipitation of overexpressed CRTC3 was monitored by immunoblotting using anti-FLAG. CRTC3 antibodies raised in sheep S276D anti-CRTC3 (3 – 13), S277D anti-CRTC3 (142 – 155), and S279D anti-CRTC (pan isoform), but not S278D anti-CRTC3 (598 – 608), immunoprecipitate overexpressed CRTC3.
Cell extract protein from RAW264.7 cells (0.75 mg) was incubated with increasing amounts of S277D anti-CRTC3 (142 – 151) (bleed 2) and proteins precipitated using protein G-Sepharose. Immunoprecipitated CRCT3 was detected by immunoblotting using anti-CRTC3 (Abcam cat. Ab91654). Depletion of CRTC3 from extracts was also measured by blotting cell extracts after immunoprecipitation. Protein amounts on gel were verified by immunoblotting for tubulin. The antibody against CRTC3 S277D can immunoprecipitate endogenous CRTC3 and can completely deplete CRTC3 from the extract.