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. 2014 Feb 18;111(9):3371–3376. doi: 10.1073/pnas.1401556111

Fig. 1.

Fig. 1.

CRL7-mediated degradation of IRS1 is impaired by SV40 LT. (A) HEK293 cells were transfected with empty vector or plasmids encoding V5-tagged IRS1, myc-tagged CRL7 substrate receptor Fbw8, HA-tagged LT, and HA-tagged CUL7 binding-deficient mutant LT∆69–83 (designated ∆LT). Where indicated, cells were treated with the proteasomal inhibitor MG132 (10 µM) for 8 h. Lysates were separated by SDS/PAGE and subjected to Western blot analysis. (B) IRS1 protein concentrations in HEK293 cells subjected to protein degradation assays as described in A. n = 9; *P < 0.05, **P < 0.01. (C) IRS1 mRNA concentrations in HEK293 cells subjected to protein degradation assays as described in A. The graph depicts quantitative real-time PCR data of five independent experiments. NS, not significant. (D) Stabilization of IRS1 steady-state protein level by SV40 LT. HEK293 cells were transfected with plasmids encoding V5-tagged IRS1, myc-tagged Fbw8, HA-tagged LT, and HA-tagged ∆LT. At 24 h after transfection, proteins were labeled for 1 h with [35S]Met/Cys and chased with normal growth medium. Cell extracts were subjected to V5 IP, separated by SDS/PAGE, and visualized by autoradiography (Lower). n = 4; *P < 0.05, **P < 0.01. Data are presented as means ± SEM.