Figure 5. The DENND2 family regulates Rab9 and lysosomes. (A) A representative panel of human Rab proteins was tested against 10 nM of His6-tagged human DENND2D purified from bacteria using the GDP-releasing assay. (B) Human DENND2A, DENND2B, and DENND2C were expressed as His6-tagged protein in bacteria and then tested against a subset of Rab9-related Rabs. Errors bars show the standard error of the mean. The red line marks double the median value. (C) HeLa cells were transfected with EGFP-tagged DENND2A or Rab9A (green), fixed after 24 h, and stained with the antibodies indicated (red). DNA was stained with DAPI (blue). Inset enlargements are shown to more clearly demonstrate the relationship between Rab9 (green) and LAMP1 (red), which suggests that Rab9 is present on the lysosome membrane. (D) HeLa cells expressing EGFP-tagged Rab9 or DENND2 constructs as indicated were transfected with control, Rab9A, Rab9B, and DENND2A-D siRNA duplexes for 72 h. Western blotting with EGFP antibodies confirmed depletion of the target proteins, whereas tubulin showed that loading was equal for all samples. HeLa cells transfected with control, Rab9A and Rab9B, and DENND2A-D siRNA duplexes for 72 h were fixed, then stained for LAMP1 (red) and DAPI to detect DNA (blue). Molecular mass standards are indicated in kilodaltons. (E) HeLa cells transfected with control, Rab9A and Rab9B, and DENND2A siRNA duplexes for 72 h were fixed, then stained for MPR (green) and TGN46 (red). DNA was stained with DAPI (blue). Bars, 10 µm. (F) Fluorescence intensity for MPR staining from E was measured using ImageJ by drawing a box around the entire cell area and integrating the total signal. An equivalent area with no cell was subtracted for the background. This was performed for 24 cells, and the mean and standard error are plotted on the bar graph. A 20 × 1 µm line measurement was performed across the nuclear region where MPR staining is most clustered. The pixel intensity along the line is plotted in the graph for control, DENND2A, and Rab9a- and Rab9b-depleted cells.
Figure 5.

The DENND2 family regulates Rab9 and lysosomes. (A) A representative panel of human Rab proteins was tested against 10 nM of His6-tagged human DENND2D purified from bacteria using the GDP-releasing assay. (B) Human DENND2A, DENND2B, and DENND2C were expressed as His6-tagged protein in bacteria and then tested against a subset of Rab9-related Rabs. Errors bars show the standard error of the mean. The red line marks double the median value. (C) HeLa cells were transfected with EGFP-tagged DENND2A or Rab9A (green), fixed after 24 h, and stained with the antibodies indicated (red). DNA was stained with DAPI (blue). Inset enlargements are shown to more clearly demonstrate the relationship between Rab9 (green) and LAMP1 (red), which suggests that Rab9 is present on the lysosome membrane. (D) HeLa cells expressing EGFP-tagged Rab9 or DENND2 constructs as indicated were transfected with control, Rab9A, Rab9B, and DENND2A-D siRNA duplexes for 72 h. Western blotting with EGFP antibodies confirmed depletion of the target proteins, whereas tubulin showed that loading was equal for all samples. HeLa cells transfected with control, Rab9A and Rab9B, and DENND2A-D siRNA duplexes for 72 h were fixed, then stained for LAMP1 (red) and DAPI to detect DNA (blue). Molecular mass standards are indicated in kilodaltons. (E) HeLa cells transfected with control, Rab9A and Rab9B, and DENND2A siRNA duplexes for 72 h were fixed, then stained for MPR (green) and TGN46 (red). DNA was stained with DAPI (blue). Bars, 10 µm. (F) Fluorescence intensity for MPR staining from E was measured using ImageJ by drawing a box around the entire cell area and integrating the total signal. An equivalent area with no cell was subtracted for the background. This was performed for 24 cells, and the mean and standard error are plotted on the bar graph. A 20 × 1 µm line measurement was performed across the nuclear region where MPR staining is most clustered. The pixel intensity along the line is plotted in the graph for control, DENND2A, and Rab9a- and Rab9b-depleted cells.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal