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. 2020 May 26;117(23):13105–13116. doi: 10.1073/pnas.1917906117

Fig. 7.

Fig. 7.

Rab10 and AS160 regulate DOPr trafficking. (A) HEK293 cells were transiently transfected with FLAG-DOPr alone or with HA-Rab10 for 48 h. Cell surface expression was measured by ELISA with FLAG-specific rabbit polyclonal and alkaline phosphatase-conjugated goat anti-rabbit antibodies. Results are shown as a percentage of cell surface FLAG-DOPr expression when HA-Rab10 is transfected compared with FLAG-DOPr alone set at 100% (n = 4, **P = 0.0100, two-tailed Student’s t test). (B–F) HEK293 cells stably expressing FLAG-DOPr were transfected with a control DsiRNA (DsiCTRL), (B–D) DsiRNAs targeting the exon 6 or 3 of the human Rab10 gene (DsiRNA-Rab10-13.2 or DsiRNA-Rab10-13.3, respectively), or (E and F) DsiRNAs targeting the exon 3 or 15 of the human AS160 gene (DsiRNA-AS160-13.1 or DsiRNA-AS160-13.2, respectively) for 72 h along with HA-Rab10 for 48 h. (B and E) Cell surface expression of FLAG-DOPr was measured as described above. Results are shown as a percentage of cell surface receptor expression when DsiRNAs targeting Rab10 or AS160 are transfected compared with the DsiCTRL condition set at 100% (B: n = 5, ****P < 0.0001; E: n = 6, **P = 0.0076 and ****P < 0.0001; one-way ANOVA with Dunnett’s multiple-comparisons test). (C) Total cell lysates were immunoblotted with a specific mouse monoclonal anti-Rab10 antibody to assess the effect of Rab10 DsiRNAs on total expression of Rab10. (F) Following RNA extraction, RT-PCRs were performed to assess the effect of AS160 DsiRNAs on total expression of AS160. Primers used are listed in SI Appendix, Table S5. Densitometry was performed using ImageJ software to quantify relative expression of Rab10 or AS160 normalized with GAPDH expression, and results were analyzed using one-way ANOVA with Dunnett’s multiple-comparisons test (C: n = 3, ***P = 0.0003 and φφφP = 0.0001; F: n = 4, ****P < 0.0001). IB, immunoblotting. (D) Cells were stimulated with SNC80 (1 µM) for up to 60 min, and cell surface expression of FLAG-DOPr was measured by ELISA as mentioned above (n = 4, *P = 0.0285, **P = 0.0017 and ***P = 0.0001; two-way ANOVA with Dunnett’s multiple-comparisons test).