Fig. 5.

TNPO1, Rab8 and f-CTS form a ternary complex. All cell lysates were from HEK293T cells. (A) f-CTS directly interacted with the GDP-locked Rab8 mutant (Rab8-TN). Bead-immobilized GST–f-CTS or GST was incubated with purified His–Rab8-wt, His–Rab8-QL and His–Rab8-TN. The protein pulled down was blotted with an anti-Rab8 antibody. (B) The interaction between f-CTS and TNPO1 should be direct. Bead-immobilized GST–f-CTS specifically pulled down TNPO1 from rabbit reticulocyte lysate, which contains endogenous TNPO1 but not Rab8 (Fig. S4B). (C) Rab8-TN indirectly interacted with TNPO1 through fibrocystin. Bead-immobilized GST–Rab8 fusion proteins were incubated with cell lysate expressing CD8a–GFP or CFF–GFP, and the material pulled down was blotted for TNPO1 and GFP chimeras. * indicates the specific protein band. (D) Rab8-TN promoted the interaction between fibrocystin and TNPO1. The cell lysates co-expressing HA–TNPO1, CFF–Myc and one of the following chimeras, GFP–Rab8-wt, GFP–Rab8-QL, GFP–Rab8-TN and GFP, was subjected to immunoprecipitation using anti-Myc antibody and co-immunoprecipitated TNPO1 and GFP chimeras were blotted. (E) Overexpression of the Rab8 GDP-locked mutant reduced the ciliary localization of fibrocystin. CPIR values of CFFΔC–Myc in ciliated RPE1 cells co-expressing CFFΔC–Myc and one of the following chimeras, GFP–Rab8-wt, GFP–Rab8-QL, GFP–Rab8-TN and GFP. The mean value is indicated at the top of each column. n=25. Error bars are s.e.m. P-values (t-test) of selected pairs are denoted. In all gel blots, numbers at the right indicate the molecular weight markers in kDa.