Fig. 3. TBC1D9 recruitment to invading GAS requires its Ca²⁺-binding site and UBR.

a, b TBC1D9 recruitment to GAS. HeLa cells expressing GFP-TBC1D9 and mCherry-LC3 were infected with GAS, fixed, and stained with DAPI. a Representative confocal images of GFP-TBC1D9 localization in uninfected or GAS-infected cells and b the time course of TBC1D9, ubiquitin, and LC3 recruitment during GAS infection. c HeLa cells were infected with GAS WT or the Δslo mutant for 4 h, fixed, and immunostained for TBC1D9. The percentage of TBC1D9-positive GAS-infected cells is shown. d WT or ATG5-KO cells expressing GFP-TBC1D9 were infected with for 4 h, and the percentage of TBC1D9-positive GAS-infected cells is shown. e Domain organization and deletion mutants of TBC1D9. f Localization of GFP-TBC1D9 deletion mutants during GAS infection. g Beads coated with GST, GST-ubiquitin, or ubiquitin-like proteins were incubated with lysate from cells expressing GFP-TBC1D9, followed by immunoblot with an antibody against GFP. h, i HeLa cells infected with GAS WT or the Δslo mutant were stained with TBC1D9 and primary antibodies against Lys63-specific ubiquitin to assess TBC1D9-ubiquitin binding by Duolink PLA. h Representative confocal micrographs and i quantification of the PLA signal per cell. Dots (red) indicate TBC1D9-ubiquitin complexes. Scale bars, 10 μm. j GST pulldown assay with indicated TBC1D9 construct and GST as a control. GST-fusion proteins were incubated with purchased diubiquitins linked by Lys48 or Lys63 (Boston Biochem) and analyzed by immunoblotting with antiubiquitin antibody. k, l HeLa cells expressing EmGFP-TBC1D9 constructs were infected with GAS, fixed, and stained with primary antibodies against anti-GFP and anti-Lys63-specific ubiquitin to assess TBC1D9-ubiquitin binding by Duolink PLA. k Representative confocal micrographs and l quantification of the PLA signal per cell. Data in (b) (n > 200 cells per condition), (c) (n > 200 cells), (d) (n > 200 cells), (i) (n > 20 cells per condition), and (l) (n > 20 cells per condition) represent the mean ± SEM of three independent experiments. P values calculated by two-tailed Student’s t test.