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. 2021 Feb 19;220(4):e202010048. doi: 10.1083/jcb.202010048

Figure 2.

Figure 2.

TBC1D5 binds retromer and LC3 independently. (A) Schematic representation of human TBC1D5, with black fonts indicating the predicted LIR motifs and blue fonts indicating the identified Ins motifs. (B) Pull-down assay to assess binding of GST-TBC1D5 and endogenous LC3B-II. HEK293T cells were transfected with GST-tagged TBC1D5-WT, TBC1D5-ΔLIR1, TBC1D5-ΔLIR2, TBC1D5-ΔLIR3, or TBC1D5-ΔLIR4 for 24 h. Cells were starved for 4 h in EBSS in the presence of 100 nM BafA1 (E+B). Control cells (vector) were transfected with empty vector. One tenth of the cell lysate was prepared as input, and the rest was used for pull-down with GST resin, followed by immunoblotting using antibodies against GST-TBC1D5 and LC3B. Representative images from one of three independent experiments are shown. (C) Confocal imaging for GST-TBC1D5 (red) and GFP-LC3 (green) immunofluorescence staining in HeLa cells stabling expressing GFP-LC3. TBC1D5 WT, LIR deletions, or empty vector was transiently transfected for 24 h, followed by starvation for 4 h. Representative images from one of two independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficient for TBC1D5 and LC3 colocalization. Results for individual cells are plotted, along with the mean and SD for each group (n = 30); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (D) Immunoprecipitation of endogenous VPS35 and LC3B from HEK293T cells transfected with HA-YFP tagged TBC1D5. WT, ATG5-KO, or ATG7-KO HEK293T cells were transfected with a HA-YFP-TBC1D5 plasmid, followed by starvation for 4 h (E+B). One tenth of the cell lysate was prepared as input, and the rest was used for immunoprecipitation with anti-HA antibody, followed by immunoblotting with antibodies against VPS35 and LC3B. Representative images from one of three independent experiments are shown. Column chart represents the relative expression of VPS35 to HA-YFP-TBC1D5. Band intensities were quantified with ImageJ and normalized to their respective control group (n = 3). Data are presented as means ± SD, and one-way ANOVA by Tukey’s HSD test or t test was used for data analysis. (E) Immunoprecipitation (IP) of endogenous VPS35 and LC3B from HEK293T cell lysates. WT, ATG5-KO, or ATG7-KO HEK293T cells were starved for 4 h in EBSS in the presence of 100 nM BafA1 (E+B). One tenth of the cell lysate was prepared as input, and the rest was used for immunoprecipitation with anti-TBC1D5 antibodies, followed by immunoblotting (IB) with antibodies against VPS35 and LC3B. Representative images from one of three independent experiments are shown. Column chart represents the relative expression of VPS35 to TBC1D5. Band intensities were quantified with ImageJ and normalized to their respective control group (n = 3). Data are presented as means ± SD, and one-way ANOVA by Tukey’s HSD test or t test was used for data analysis. (F) Model showing the interaction between TBC1D5 and retromer (VPS35/VPS26/VPS29) or LC3-II. HSD, honestly significant difference.