TMED9 knockdown blocks RESET and consequent anti-GFP-antibody uptake in YFP-PrP * NRK cells.

(A) Immunofluorescence images of TMED9 staining in YFP-PrP * NRK cells that were treated with scrambled control siRNA (CTL) or siRNA against TMED9 (siTMED9). With our TMED9-knockdown protocol, the majority of siTMED9-treated YFP-PrP * NRK cells demonstrated TMED9 knock-down. A minor population (~30%–35%) of the siTMED9-treated cells did not demonstrate knock-down of TMED9. (B–D) Time-lapse images of cells collected immediately after the addition of TG, Alexa Fluor 647 (AF647)-conjugated rabbit anti-GFP antibodies, and bafilomycin A1. There is a faint ambient signal from the AF647-conjugated antibody in the medium that can be enhanced by increasing the gain. Scale bar represents 10 µm. (B) YFP-PrP * NRK cell that was treated with scrambled control siRNA (CTL). (C) Parental untransfected NRK cell that was treated with scrambled control siRNA (CTL). The outline of the cell has been hand-traced in cyan. (D) YFP-PrP * NRK cells that were treated with siRNA against TMED9 (siTMED9). A field of view that contained one cell that displayed the phenotype of complete TMED9 knockdown versus one cell with incomplete knockdown was selected for comparison. This panel is associated with S2 Video.

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