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. Author manuscript; available in PMC: 2018 Sep 7.
Published in final edited form as: Mol Cell. 2017 Sep 7;67(5):770–782.e6. doi: 10.1016/j.molcel.2017.07.017

Figure 2. CLOCK-BMAL1 becomes fully incorporated into the PER complex.

Figure 2

(A) BN-APAGE immunoblots showing circadian profiles of liver nuclear protein complexes containing BMAL1 (top) or CLOCK (bottom) at the indicated circadian times. Arrow, position of PER complex. Asterisk, non-specific. Loss of CLOCK signal in Bmal1−/− (Bmal1 KO) suggests instability of CLOCK monomer. See Figure S2.

(B) Quantitative incorporation of CLOCK-BMAL1 at the peak of the repression phase. BN-APAGE immunoblots of nuclear extracts from livers collected at CT19 from three wildtype mice, probed for the proteins indicated at bottom. Arrow, trace ~750-kDa complex containing CLOCK-BMAL1. Three biological replicates shown.

(C) High molecular-weight complex containing CLOCK-BMAL1 is the PER complex. Mouse liver nuclear extracts (CT19) were immunodepleted with control IgG or anti-PER2 antibody (top), or IgG and anti-CRY1 antibody (bottom), and the depleted (unbound) fraction was analyzed by BN-APAGE. Immunoblots of the gels were probed for proteins indicated at bottom.

(D) Genetic analysis of incorporation of CLOCK-BMAL1 into the PER complex. Top, BN-APAGE immunoblot of liver nuclear extracts (CT18) from wildtype (WT) or mutants null for the circadian clock genes indicated at the top (KO) probed for CLOCK. Middle and bottom, SDS-PAGE immunoblots showing CLOCK expression and SAP155 loading control, respectively.