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Fig. 3. TPR1 downregulation obstructs vesicular transport of the VSVG^ts045-GFP cargo protein in vivo. (A) Decrease of TPR1 expression levels in HEK293 cells by specific siRNA. Cells were mock transfected, treated with siRNA targeting human TPR1 or with mutated or control siRNA. Equal protein loading is shown by an antibody specific for GAPDH. (B) Time-course of intracellular VSVG^ts045-GFP protein trafficking in cells treated with control siRNA or siRNA targeted against human TPR1. Localization of VSVG^ts045-GFP and the Golgi marker GM130 was assessed after 0, 15 and 60 minutes. Bar, 10 µm. (C) Resistance of VSVG^ts045-GFP protein against EndoH_f digestion in cells treated with control siRNA or siRNA specific for TPR1 after 0, 15 and 60 minutes at 32°C. The markers `low' and `high' refer to the endoplasmic reticulum form of VSVG^ts045-GFP from cells that have been kept at 40°C after and before EndoH_f treatment (Balch and Keller, 1986). Asterisks indicate two EndoH_f-resistant bands, a hallmark of arrival in the medial-Golgi compartment (Schwaninger et al., 1992). Presented is a typical result of an experiment performed three times. (D) EndoH_f resistancy of VSVG after 0, 15 and 60 minutes in cells treated with control siRNA or siRNA specific for TPR1. Quantification was based on the areas in each blot that correspond to the two bands marked with asterisks in C and is represented as a percentage of the total signal measured. Bars, ± s.e.m.

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