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. 2019 Sep 9;129(10):4393–4407. doi: 10.1172/JCI129107

Figure 2. CCDC8 localizes on and recruits OBSL1 and CUL7 to the plasma membrane.

Figure 2

(A) Schematic representation of the domain structure and knockin epitope tagging of endogenous CUL7 and CCDC8 to generate 16×MYC-CUL7 and CCDC8-21×FLAG double-knockin (DKI) U2OS cells. (B) Validation of DKI U2OS cells. Cell lysates from DKI and parental U2OS cells were subjected to anti-FLAG immunoprecipitation (IP) and Western blot. (C) Immunostaining of DKI and parental U2OS cells with antibodies specific for MYC, FLAG, or plasma membrane marker Na+/K+-ATPase demonstrates the colocalization of endogenous (endo.) CCDC8 and CUL7 on the plasma membrane. Scale bars: 10 μm. (D) U2OS cells transfected with shRNA targeting CCDC8 were fractionated by centrifugation, and total lysate (Lys), cytoplasm (Cyto), and membrane (Mem) fractions were analyzed by Western blot with antibodies recognizing the indicated proteins. CUL7 and CUL9 were recognized by the same monoclonal antibody. (E) Induced overexpression of CCDC8-mKate localized CUL7-mTagBFP and OBSL1-EGFP onto the plasma membrane. Dox, doxycycline. Scale bars: 10 μm.