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TECPR2 directly interacts with SNX17 and regulates the association of SNX17 with Rab5-positive endosomes. A Live-cell imaging of HeLa cells co-expressing GFP-TECPR2 (green), Halo-SNX17 (gray) and untagged-Rab5 (WT). Endocytic uptake of active Itgβ1 (magenta) was performed as described in Fig. 6E (see Supplementary Movie 9). Arrowheads indicate colocalized pixels in all three channels. Scale bar: 5 µm. B GST and GST-SNX17 were used to pull down FLAG-TECPR2 expressed in HEK293T cells. The FLAG-tagged proteins were detected by immunoblotting and GST proteins using Ponceau S staining. C GST and GST-SNX17 were used to pull down the MBP or MBP-TECPR2_TECPR (WT) and (R1336W) variant. The MBP-tagged proteins were detected by immunoblotting and GST proteins using Ponceau S staining. Densitometric analysis of TECPR2_TECPR protein, normalized to the input and direct pulldown, is shown. D HEK293T cell lysates expressing either FLAG-TECPR2 (WT) or the (R1336W) variant were immunoprecipitated with anti-FLAG and immunoblotted (IB) with the indicated antibodies. Densitometric analysis of the indicated co-immunoprecipitated proteins from HEK293T cell lysates, normalized to the input and direct IP, is shown. E Live-cell imaging of HeLa cells treated with indicated siRNA and co-expressing Halo-SNX17 (magenta) and GFP-Rab5 (WT) (green). Arrowheads indicate endosomes positive for Halo-SNX17 and GFP-Rab5. Scale bars: 10 µm (main); 5 µm (inset). The line-scan analysis shows the fluorescence intensity of Halo-SNX17 and GFP-Rab5 across the line drawn on the indicated endosome normalized to the background (see Supplementary Movie 10). F Quantification of the Pearson’s colocalization coefficient of Halo-SNX17 and GFP-Rab5 (WT) in HeLa cells treated with indicated siRNA. The values are represented as described for Fig. 2B. The error bar represents mean ± SD from three independent experiments (****p < 0.0001; unpaired two-tailed Student’s t-test). G GST and GST-SNX17 were used to pull down control and TECPR2-depleted HEK293T lysates. Rab5 and TECPR2 were detected by immunblotting and GST proteins using Ponceau S staining. Densitometric analysis of Rab5, normalized to the input and direct pulldown, is shown. A non-specific band detected by the anti-TECPR2 antibody is marked with an asterisk (*). H HeLa cells were treated with indicated siRNA and incubated with ferrofluid (FF) for 30 min at 37 °C. The FF-containing compartments were purified and IB for the presence of indicated proteins. SE = Short Exposure; LE = Long Exposure. I Densitometric analysis of SNX17, Rab5 and EEA1 upon TECPR2 depletion, normalized to the input and control siRNA. The data shown is the mean ± SD from three independent experiments, such as that shown in the panel (H). Experiments are color-coded, where each dot represents the mean value (***p = 0.0004 for SNX17 and 0.0002 for Rab5; ns=0.0727 for EEA1; unpaired two-tailed Student’s t-test). Source data are provided as a Source Data file.
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