FIGURE 8.

Analysis of TRPC6 trafficking to the cell surface in response to H₂O₂. A, biotinylation of TRPC6 protein in HEK293T cells transfected with TRPC6 (T6) or empty vector (Mock) with and without H₂O₂ stimulation. L, a protein ladder; T6+UT, TRPC6-expressing cells without H₂O₂ treatment; T6+H₂O₂, TRPC6-expressing cells treated with 10 μm H₂O₂ for 3 min; Mock+H₂O₂, empty vector-transfected cells treated with 10 μm H₂O₂ for 3 min; Inputs, the whole-cell lysates (unbiotinylated), ∼1:10 of the lysates used for biotinylation. B, quantitative analysis of the cell surface TRPC6 protein with and without H₂O₂ stimulation. Data were expressed as percentages of integrated optical density of TRPC6 immunoblots in biotinylated fractions to the corresponding inputs, calculated by (integrated optical density of biotinylated TRPC6 blot/(integrated optical density of TRPC6 blot in input × 10)) × 100. n indicates the number of independent experiments. The optical density of immunoblots was measured with a software provided by AlphaEaseFC Imaging System (Alpha Innotech). Asterisk indicates significant difference compared with T6+UT. C, representative confocal microscopy photographs, showing TRPC6 trafficking to the region of the plasma membrane in response to 10 μm H₂O₂ stimulation in the cells transfected with TRPC6-EGFP (T6-EGFP), but not in the cells transfected with EGFP alone. Images were consecutively captured before application, immediately, 2, and 4 min after application of H₂O₂ (labeled with 0, 1′, 3′, and 5′, respectively). The duration of scanning one image was ∼1 min. The rainbow bars on the right indicates the fluorescence intensity in arbitrary units. Arrows indicate the sites of an increase in fluorescence intensity compared with before application of H₂O₂ (time 0). The horizontal bars in the left panels represent 20 μm. D, time course changes in fluorescence intensity on the cell surface in response to H₂O₂ in TRPC6-EGFP (T6-EGFP) or EGFP alone (EGFP) transfected cells. Data were expressed as percent changes from the values before application of H₂O₂ (time 0). The fluorescence intensity was measured using the software SymPhoTime (version 5.0). Asterisk indicates significant difference between T6-EGFP and EGFP at the corresponding time point. n indicates the number of analyzed cells from four sets of transfection. E, percent changes in fluorescence intensity on the cell surface 3 min after application of AVP (100 nm) in HEK293 cells cotransfected with EGFP or TRPC6-EGFP (T6-EGFP) and V1R with and without pretreatment of PEG-catalase (250 units/ml for 30 min). See D for the approach for data analysis. Asterisk indicates significant difference between the indicated groups. n indicates the number of analyzed cells from four sets of transfection.