Figure 2. FNIP interacts with SCF^β-TRCP.

A. Immunoblot (IB) analysis of whole cell lysates (WCL) and Flag-immunoprecipitates (IP) derived from 293T cells transfected with Flag-FNIP2 and empty vector (EV), Myc-Cullin1, 2, 3, 4A, or 5, as indicated. At 24 h post transfection, cells were treated with 15 μM MG132 for 12 h before harvesting. B-C. IB analysis of WCL and Flag-IP derived from 293T cells transfected with Flag-FNIP2 and Myc-SKP1 (B) or Myc-RBX1 (C) as indicated. At 24 h post transfection, cells were treated with 15 μM MG132 for 12 h before harvesting. D. IB analysis of WCL derived from HeLa cells infected with lentiviral shRNA constructs against GFP and Cullin1 (four independent shRNAs namely Cullin1-A, B, C, or D) followed by selection with 1 μg/mL puromycin for 72 h to eliminate non-infected cells. E. IB analysis of WCL derived from HeLa cells infected with lentiviral shRNA constructs against GFP, β-TRCP1, β-TRCP2, β-TRCP1+2 (shRNA against both β-TRCP1 and β-TRCP2 paralogs), and SKP2 (three independent shRNAs namely SKP2-A, B, and C) followed by selection with 1 μg/mL puromycin for 72 h to eliminate non-infected cells. F. IB analysis of WCL and HA-IP derived from HeLa cells transfected with HA-FNIP2 along with empty vector (EV), Flag-β-TRCP1, or Flag-SKP2. At 24 h post transfection, cells were treated with 15 μM MG132 for 12 h before harvesting. G. IB analysis of WCL and FNIP2-IP derived from HeLa cells treated with MG132 for 12 h before harvesting. Endogenous interaction between FNIP2 and β-TRCP1 was confirmed. Input, 5% WCL. H. IB analysis of WCL and Flag-IP derived from HeLa cells transfected with Flag-FNIP2 along with empty vector (EV), Myc-β-TRCP1, or Myc-β-TRCP1-R474A. At 24 h post transfection, cells were treated with 15 μM MG132 for 12 h before harvesting.