Figure 2.

STUB1 preferably interacts with BMAL1 over CLOCK. A and B, STUB1 co-immunoprecipitates BMAL1 (A) and BMAL1 co-immunoprecipitates STUB1 (B). HEK293T cells were transfected with pcDNA3.1, Myc-STUB1, or FLAG-BMAL1 plasmid using PEI transfection reagent for 48 h. STUB1 or BMAL1 was immunoprecipitated (IP) with Myc or FLAG antibodies, respectively. C, STUB1 interacts with BMAL1 endogenously. Endogenous BMAL1 in cell lysate from HEK293T cells was immunoprecipitated with an anti-BMAL1 antibody. IgG was used as a control. D, STUB1 interacts with CLOCK. HEK293T cells were transfected with HA-CLOCK plasmid and then split into two plates for the transfection of control or Myc-STUB1 plasmid for 48 h, respectively. STUB1 was immunoprecipitated with an anti-Myc antibody. HSP70 and HSP90β were used as positive controls for their interaction with STUB1. E, STUB1 preferably interacts with BMAL1 over CLOCK. HEK293T cells were co-transfected with HA-CLOCK and Myc-STUB1 plasmids and then split into two plates for the transfection of control or FLAG-BMAL1 plasmid, respectively. STUB1 was immunoprecipitated with anti-Myc magnetic beads. F, BMAL1 knockdown enhances the interaction between STUB1 and CLOCK. HEK293T cells were co-transfected with HA-CLOCK and Myc-STUB1 plasmids and then split into two plates for the transfection of control or shBMAL1 plasmid, respectively. CLOCK was immunoprecipitated with anti-HA magnetic beads. The cell lysates and immunoprecipitates in A–F were immunoblotted with the indicated antibodies.