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. 2017 Sep 4;6:e24994. doi: 10.7554/eLife.24994

Figure 5. COPS7B stabilizes the essential clock protein BMAL1.

(a–b) Coimmunoprecipitation assay of HEK293T cells transfected with plasmids expressing FLAG epitope-tagged clock proteins (BMAL1-flag, PER2-flag and CRY1-flag). *Unspecific background bands. (a) immunoprecipitated with antibodies against FLAG or (b) against endogenous COPS7B. (c) Pulse-chase analysis of BMAL1-flag protein stability in absence (left; black diamonds in graph below) or presence (right; grey squares in graph below) of siRNAs targeting COPS7B in HEK293T cells. All cells were incubated with 35S-labelled methionine-cysteine for 1 hr, and chased with excess unlabeled cysteine for 0 hr, 2 hr, 4 hr, or 6 hr before immunoprecipitation. Upper panel, representative radioblot; lower panel, quantification from 3 experiments ± s.d., expressed as percentage of labeled immunoprecipitated protein (relative to 0 hr) at indicated time. *p=0.04999, Student T-test. (d) Immunoprecipitation experiments between BMAL1 and COPS7B performed in U2OS cells. See also Figure 5—figure supplement 1 for negative control experiments upon untransfected cells; see Figure 5—figure supplement 2 for pulse-chase analysis of PER2-flag and CRY1-flag protein stability in the presence or absence of siRNAs targeting COPS7B.

Figure 5.

Figure 5—figure supplement 1. Negative control immunoprecipitation experiments identical to those described in Figure 5a,b, but using untransfected HEK293T cells as a substrate.

Figure 5—figure supplement 1.

Coimmunoprecipitation assay of untransfected HEK293T cells. (left) immunoprecipitated with antibodies against flag and probed with anti COPS7B and (right) immunoprecipitated with antibodies against endogenous COPS7B and probed with anti-FLAG. * background bands of size unrelated to the proteins in question.
Figure 5—figure supplement 2. Pulse-chase analysis of PER2 (a) and CRY1 (b) protein half-lives in absence (left-control lanes; black diamonds in graph below) or presence (right lanes; grey squares in graph below) of siRNAs targeting COPS7B.

Figure 5—figure supplement 2.

HEK293T cells were transfected either with FLAG epitope-tagged clock proteins (PER2-flag and CRY1-flag) or FLAG epitope-tagged clock proteins plus siRNA targeting COPS7B, incubated with 35S-labelled methionine-cysteine for 1 hr, and chased with excess unlabeled methionine-cysteine for the indicated times (x-axis, 0 hr, 2 hr, 4 hr, 6 hr) before immunoprecipitation. Upper panel, autoradiography of representative immunoprecipitated samples; lower panel, quantification from 3 experiments,±s.d. Y-axis, percentage of labeled immunoprecipitated protein (relative to 0 hr) at indicated time.