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Rab5 recruits TECPR2 on early endosomes, a process disrupted in the HSAN-associated TECPR2 (R1336W) variant. A Representative confocal micrographs of HeLa cells transfected with GFP-TECPR2 and immunostained for endogenous Rab5 and EEA1. The line profiles indicate fluorescence intensity along the yellow line for both channels. Arrowheads in the insets denote the colocalized pixels. Scale bars: 10 µm (main); 5 µm (inset). B Quantification of the Pearson’s colocalization coefficient for GFP-TECPR2 with the indicated markers is shown. n denotes the total number of cells analyzed, and the error bar represents the mean ± SD from three independent experiments. Experiments are color-coded, and each dot represents the individual data points (****p < 0.0001; unpaired two-tailed Student’s t-test). C HeLa cells were treated with the indicated siRNA and incubated with ferrofluid (FF) for 30 min at 37 °C, followed by a chase in complete media for the indicated time. The FF-containing compartments were purified at the indicated time points, and immunoblotted (IB) for the indicated proteins. SE = Short Exposure; LE = Long Exposure. Densitometric analysis of TECPR2, normalized to the input, is shown. D Representative confocal micrographs of HeLa cells co-transfected with GFP-TECPR2 and RFP-Rab5 (WT) and mutants. Arrowheads in the insets denote the colocalized pixels. Scale bars: 10 µm (main); 5 µm (inset). E Representative confocal micrographs of HeLa cells co-transfected with either GFP or GFP-TECPR2 (WT and R1336W) with Rab5 (Q79L and S34N)-Mito-HA and immunostained with anti-HA antibodies. Scale bars: 10 μm. F Quantification of the percentage of cells in which GFP-TECPR2 (WT and R1336W) was re-localized to mitochondria. A total of 16 cells were analyzed per experiment for each sample. The data shown is mean ± SD from three independent experiments. Experiments are color-coded, where each dot represents the mean value (****p < 0.0001; unpaired two-tailed Student’s t-test). G Representative confocal micrographs of Liss-Rhodamine-labeled GUVs incubated with purified TECPR2_TECPR (WT) and TECPR2_TECPR (R1336W) in the absence or presence of purified GTP-loaded Rab5. The GUVs were immunostained with anti-TECPR2 antibody (green). Scale bars: 10 μm. H Quantification of the fluorescence intensity of TECPR2_TECPR (WT/R1336W) per perimeter of GUV in the absence or presence of GTP-loaded Rab5. The values are represented as described for Fig. 2B, and n denotes the total number of GUVs analyzed. The data shown is mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). I Representative confocal micrographs showing FLAG-TECPR2_TECPR localization in HeLa cells treated with indicated siRNA. The cells were immunostained with anti-FLAG and anti-Rab5 antibodies. Arrowheads in the insets denote the colocalized pixels. Scale bars: 10 µm (main); 5 µm (inset). Source data are provided as a Source Data file.
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