Fig. 5: LRBs and COP1 are both required for CRY2 ubiquitination.
From: Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases
Immunoblots showing the ubiquitination of FGFP-CRY2 in indicated genotypes. 7-day-old etiolated seedlings constitutively expressing FGFP-CRY2 in indicated genotypes were pretreated with MG132 and kept in the dark or exposed to 30 μmol m−2 s−1 of blue light for 5, 10, and 15 min. The blue light treated samples (5, 10, and 15 min) were pooled together while performing immunoprecipitation for (a–e). a–c Total ubiquitinated proteins were purified by TUBE2-conjugated beads. Immunoprecipitated proteins were analyzed by immunoblots probed with anti-ubiquitin antibody (α-Ubq) or anti-CRY2 antibody (α-CRY2). The extent of CRY2 ubiquitination was shown below the immunoblots, calculated as [CRY2-Ubq intensity]IP/[Ubq intensity]IP. d–e FGFP-CRY2 proteins were purified with GFP-trap beads. Immunoprecipitated proteins were analyzed by immunoblots probed with anti-ubiquitin antibody (α-Ubq), anti-Flag antibody (α-Flag) or anti-Myc antibody (α-Myc) for detecting epitope-tagged proteins. The extent of CRY2 ubiquitination is calculated as [CRY2-Ubq intensity]IP/[Flag intensity]IP with the short exposure immunoblots and shown below the immunoblots. S. exp or L. exp: short or long chemiluminescence exposures of immunoblots. D, dark treatment; B, blue light treatment. The above experiments were repeated at least twice with similar results.
