Fig. 2.

Brg1 interacts with FBW7 in a CKIδ-mediated phosphorylation dependent manner. a Sequence alignment of Brg1 with the phospho-degron sequences recognized by FBW7. The putative FBW7 phospho-degron sequence present in Brg1 is conserved across different species. b IB analysis of WCLs and IPs derived from 293T cells transfected with HA-FBW7 together with the indicated Flag-Brg1 constructs. c IB analysis showing the enhanced binding of HA-FBW7 with bacterially purified GST-Brg1 (1–104aa) proteins after incubation with CK1 kinase in vitro. GST was used as a negative control. d IB analysis of WCLs and IPs derived from 293T cells transfected with HA-FBW7 and Flag-Brg1. Where indicated, the CK1 inhibitor IC261 (20 μM) was added for 8 h before harvesting for IB analysis. DMSO was used as a negative control. e IB analysis of WCLs derived from MKN45 cells infected with the indicated lentiviral shRNA-CK1δ constructs. f IB analysis of WCLs derived from 293T cells transfected with the indicated Flag-Brg1 and HA-FBW7 vectors in the presence or absence of Myc-CK1δ. GFP was used as a negative control. g, h 293T cells were transfected with the indicated Flag-Brg1 constructs together with HA–FBW7 and Myc-CK1δ plasmids. Twenty hours after transfection, cells were split into 60-mm dishes. After another 20 h, cells were treated with 20 μg/ml CHX. i IB analysis of WCLs derived from 293T cells transfected with Flag-Brg1 and the indicated HA-FBW7 vectors. Where indicated, MG132 (10 μM) was treated for 10 h. j IB analysis of His tag pull-down and WCLs derived from 293 cells transfected with indicated Flag-Brg1 constructs together with the HA-FBW7, Myc-CK1δ, and His-Ub plasmids. 20 h after transfection, cells were treated with MG132 for 10 h before cell collection. His tag pull-down was then performed. Ni-NTA: nickel-nitrilotriacetic acid, Ub: ubiquitin