Fig. 7. mPER2 proteins inhibit mCRY1 and hCRY2 ubiquitylation. (A) Effect of mPER2 on the ubiquitylation of CRY proteins in COS7 cells transiently expressing mouse CRY1, hCRY2, mPER2(full)–GFP and/or FLAG-tagged ubiquitin (as indicated) and cultured in the presence of MG132. CRY proteins were immunoprecipitated with anti-CRY1 (TD1119) or anti-CRY2 (TD2206) antibodies and precipitates were examined by western blot analysis using an anti-FLAG (top) antibody. Expression of mPER2–GFP was confirmed by western blot analysis of total cell lysates using anti-mPER2 antibodies (bottom). (B) As (A), except that another antiserum against CRY2 (RY1083) was used for immunoprecipitation and one cell lysate was prepared from cells grown in the absence of MG132. Note the low levels of ubiquitylated mPER2 in the latter sample. (C) Cell-free degradation of transiently expressed hCRY2. COS7 cell lysates were incubated at room temperature for the indicated time in the absence or presence of MG132 (50 µM) or lactacystin (50 µM). hCRY2 was visualized by western blot analysis using anti-CRY2 antibodies. Anti-actine antibodies were used as a loading control.