Fig 1: Rab7 Cycling Is Not Required for Trafficking of Cellular Retromer Cargos(A) HeLa-tTA cells stably expressing empty vector or expressing DN or CA Rab7A plus Rab7B in the absence of doxycycline were transfected with a plasmid expressing the CD8-CIMPR fusion protein. After 24 h, live cells were incubated with anti-CD8 antibody for 3 h at 37°C and then fixed and stained with anti-TGN46. In the second row, cells expressing empty vector were transfected with siRNA targeting TBC1D5 24 h before transfection of the CD8-CIMPR plasmid. CD8-CIMPR, green; TGN46, red; nuclei, blue. Co-localization of CD8-CIMPR and TGN46 is pseudocolored yellow in the merged panels.(B) Pearson’s correlation coefficient for TGN46-CIMPR overlap is shown for 30 cells for each condition, presented as mean and standard deviation. Each dot represents an individual cell.(C) HeLa-tTA cells as in (A) were transfected with a plasmid expressing GFP-tagged DMT1-II. After 24 h, cells were stained with antibodies recognizing EEA1 and GFP. GFP-DMT1-II, green; EEA1, red; nuclei, blue. Co-localization of GFP-DMT1-II and EEA1 is pseudocolored yellow in the merged panels.(D) Pearson’s correlation coefficient for EEA1-DMT1-II overlap is presented as in (B).A single Z-plane is shown in all confocal images. ****p < 0.0001. See also Table S1.
Fig 2: TBC1D5 Is Required for HPV Infection and Disassembly of the Retromer-HPV Complex(A) HeLa S3 cells were transfected with non-targeting control scrambled siRNA or TBC1D5 siRNA. After 48 h, cells were mock-infected or infected with HPV16-GFP PsV at an MOI of 2. At 48 h.p.i., GFP fluorescence was measured by flow cytometry. Left panel: flow cytometry histograms. Right panel: average results and standard deviation of three independent experiments. Infectivity was normalized to infected cells treated with control siRNA, set at 100%. S, control scrambled siRNA; KD, TBC1D5 knockdown.(B) HeLa S3 cells transfected with control scrambled siRNA or TBC1D5 siRNA. After 48 h, cells were mock-infected or infected with HPV16-HcRed PsV at an MOI of 150. At 8 and 16 h.p.i., a PLA was performed with antibodies recognizing HPV L1 and EEA1.(C) Multiple images as in (B) were processed and presented as in Figure 2B.(D) HeLa S3 cells were transfected and infected as in (B). At 16 h.p.i., a PLA was performed with antibodies recognizing HPV L1 and TGN46.(E) Multiple images as in (D) were processed and presented as in Figure 2D.(F) Cells were transfected and infected as in (B). At 8 and 16 h.p.i., a PLA was performed with antibodies recognizing HPV L1 and VPS35.(G) Multiple images as in (F) were processed and presented as in Figure 2F.In all confocal images, a single Z-plane is shown. ****p < 0.0001. See also Figures S2 and S5.
Fig 3: HPV Recruits TBC1D5 to Endosomes via Retromer(A) HeLa S3 cells transfected with control siRNA or VPS35 siRNA were mock-infected or infected 48 h later with wild-type HPV16-HcRed PsV at an MOI of 150 or with HPV16 DM PsV containing the same number of encapsidated HcRed reporter plasmids. At 12 h.p.i., cells were stained with anti-EEA1 and anti-TBC1D5 antibodies, and fluorescence was visualized by confocal microscopy. TBC1D5, green; EEA1, red; nuclei, blue. Co-localization of EEA1 and TBC1D5 is pseudocolored yellow in the merged panels. These images show cells transfected with control siRNA; VPS35 KD cells are shown in Figure S6A.(B) Co-localization signal in the merged images as in (A) was quantified by ImageJ software from at least 60 cells in three independent experiments, evaluated by the Pearson’s correlation coefficient, and presented as mean and standard deviation. At least 30 cells as in Figure S6A were analyzed similarly. Each dot represents an individual cell. ****p < 0.0001; n.s., not significant.(C) HeLa S3 cells were mock-infected or infected as in (A) in the absence of transfection. At 8 and 16 h.p.i., cells were subjected to a PLA for VPS35 and EEA1 (left panels) or immunostained with anti-VPS35 (green) and anti-EEA1 (red) (right panels). Overlap between VPS35 and EEA1 staining is pseudocolored yellow in the right panels, which show 3D reconstructions. VPS35-EEA1 PLA signals are quantified in Figure S6B.(D) HeLa S3 cells were mock-infected or infected as in (A). At 12 h.p.i., cells were stained with anti-TBC1D5 and anti-VPS35 antibodies. TBC1D5, green; VPS35, red; nuclei, blue. Co-localization of VPS35 and TBC1D5 is pseudocolored yellow in the merged panels and quantified in Figure S6F.(E) HeLa S3 cells were transfected and infected as described in (A). At 12 h.p.i., extracts were prepared and processed as in the legend for Figure 3E to detect VPS35-TBC1D5 complex formation. -, mock-infected; WT, wild-type; DM, double mutant.(F) HeLa S3 cells were infected as in (A) in the absence of transfection. At 12 h.p.i., extracts were processed with GST or GST-RILP fusion protein as in Figure 3A to detect GTP-Rab7. Lanes identified as in (E). The positions of GST and RILP-GST are indicated by arrows.(G) HeLa S3 cells were transfected with scrambled siRNA (-) or siRNA targeting VPS35 (+). After 48 h, cells were infected with wild-type HPV16 PsV at an MOI of 150 or DM HPV16 PsV containing the same number of encapsidated reporter plasmids, both containing FLAG-tagged L2. At 12 h.p.i, cells were treated with cross-linker, and HPV PsV were immunoprecipitated with anti-FLAG antibody and immunoblotted with the indicated antibodies. Lanes identified as in (E).In all confocal images, except as noted in (C), a single Z-plane is shown. See also Figures S3,S4, and S6.
Fig 4: JX2 Forms a Complex with TBC1D5, Inhibits VPS35-TBC1D5 Interaction, and Causes Accumulation of Rab7-GTP(A) Clonal HeLa-tTA cells stably expressing FA (-) or JX2 (+) in the absence of doxycycline were transfected with non-targeting control scrambled siRNA (-) or TBC1D5 siRNA (+). After 48 h, lysates were pulled down with GST or GST-RILP and subjected to SDS-PAGE. Rab7 was detected by immunoblotting with an antibody that recognizes Rab7A and Rab7B. Samples not pulled down (input) were electrophoresed and immunoblotted for the indicated proteins. Actin is a loading control. The positions of GST and RILP-GST are indicated.(B) Cells as in (A) were transfected with non-targeting control scrambled siRNA (-) or siRNA targeting VPS35 or TBC1D5 (+). After 48 h, lysates were prepared, and JX2 was immunoprecipitated with anti-FLAG antibody. Samples were subjected to SDS-PAGE and immunoblotted for TBC1D5. Non-immunoprecipitated samples (input) were electrophoresed and blotted for the indicated proteins.(C) Clonal HeLa-tTA cells expressing FA (-) or JX2 (+) in the absence of doxycycline were mock-infected or infected with HPV16-GFP PsV at an MOI of 150. At 12 h.p.i., samples were collected and processed as in (B).(D) HeLa-tTA cells expressing FA (-) or JX2 (+) in the absence of doxycycline were transfected with siRNA targeting TBC1D5 or left untreated. Extracts were prepared 2 days later, immunoprecipitated with anti-FLAG antibody, and electrophoresed in parallel on two gels. The gel on the left was stained with silver. Arrowheads show the positions of novel immunoprecipitated bands in cells expressing JX2. M indicates the marker lane. The gel on the right was immunoblotted for TBC1D5 (top) or FLAG (bottom).(E) Cells as in (A) were transfected with control scrambled siRNA (-) or VPS35 siRNA (+). After 48 h, extracts were prepared and immunoprecipitated with anti-VPS35 antibody and blotted for TBC1D5 and VPS35. Non-immunoprecipitated samples (input) were processed as in (B).See also Figure S5.
Fig 5: Rab9a knockdown enhances HPV-retromer association even in cells overexpressing GDP-Rab7 despite increases GTP-Rab7 abundance.(A) HeLa S3 cells were transfected with siNC, siRab9a or siTBC1D5. At 48 h after transfection, lysates were pulled down with GST or GST-RILP (IP) and subjected to Western blot analysis together with samples not pulled down (input) using antibodies recognizing Rab7, GST, TBC1D5, or Rab9a. Similar results were obtained in two independent experiments. (B) HeLa S3 cells were transfected with siNC or siRab9a and mock-infected or infected at the MOI of ~200 with HPV16 PsV L2-3XFLAG containing the HcRed reporter plasmid. At 12 hpi, PLA was performed with antibodies recognizing EEA1 and Rab7. PLA signals are green; nuclei are blue (DAPI). Similar results were obtained in two independent experiments. (C) The fluorescence of PLA signals was determined from multiple images obtained as in (B). Each dot represents an individual cell (n>30) and black horizontal lines indicate the mean value of the analyzed population in each group. ***, P < 0.001; ****, P < 0.0001. The graph shows results of a representative experiment. Similar results were obtained in two independent experiments. (D) As in (B) except PLA was performed at 12 hpi with antibodies recognizing VPS35 and Rab7. Similar results were obtained in two independent experiments. (E) Images as in (D) were analyzed as described in (C). (F) As in (B) except HeLa S3 cells stably transduced with an empty vector or a plasmid expressing dominant negative Rab7 (Rab7 DN) were used, and PLA was performed at 8 hpi with antibodies recognizing FLAG and VPS35. Similar results were obtained in two independent experiments. (G) Images as in (F) were analyzed as described in (C). *, P < 0.05.
Supplier Page from Abcam for Anti-TBC1D5 antibody