antibody against rab7 #9367  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A-D) Confocal images of HeLa cells treated with 0.02% vehicle (DMSO), 240 nM apilimod, or 1 µM YM-201636 for 2 h followed by Proximity Ligation Assay between protrudin and VapA (A), or Rab7 and VapA (B), or Rab7 and protrudin (C), or Rab7 and Kinesin-1 (KIF5B; D). Images were generated by maximum projection of z-stacks of the PLA signal (magenta); DAPI-stained nucleus is shown in gray. Negative controls where one of the primary antibodies was missing are shown. Scale bar = 15 µm. (E-H) Quantification of the number of PLA dots per cell for each protein pair tested in A-D, as indicated in image. All experiments were repeated four independent times with matching independent experiments colour coded. Shown is the mean ± SEM. Data were analyzed using an Ordinary one-way ANOVA and Dunnett’s multiple comparisons test and p values are shown.

Article Snippet: Cells were then subjected to mouse monoclonal (DM1A) antibody against Ill-tubulin (1:100; #T9026; Sigma-Aldrich), rabbit monoclonal (D9F52) antibody against Rab7 (1:100; #9367), mouse monoclonal (E9O7E) antibody against Rab7 (1:100; #95746), mouse monoclonal (D4O1S) antibody against LAMP1 (1:100; #15665) and rabbit monoclonal (D2D11) antibody against LAMP1 (1:200; #9091; all from Cell Signaling Technology).

Techniques: Proximity Ligation Assay, Generated, Staining

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A) Confocal images of HeLa cells co-transfected with non-targeting siRNA control or siRNA oligonucleotides against protrudin, and protrudin wt -GFP or protrudin FYVE4A -GFP (gray). Cells were labeled with Dextran, Alexa 546 (magenta) and then exposed to either 0.02% vehicle (DMSO) or 240 nM apilimod for 2 h. Scale bar: full size = 20 µm, zoom insert = 5 µm. (B-D) Quantitative analysis of ER morphology based on protrudin wt -GFP or protrudin FYVE4A -GFP fluorescence in conditions shown in A: number of protrudin branches per cell (B), junctions per cell (C), and average branch length (D). (E, F) Quantification of number of PLA dots between Rab7 and protrudin (E) and Rab7 and VapA (F) in control silenced and protrudin-silenced cells with and without apilimod. (G, H) Quantification of PLA dots between Rab7 and protrudin (G) and Rab7-VapA (H) in cells treated with vehicle, apilimod, or Vps34-IN to abate PtdIns(3)P levels. (I) Quantification of PLA dots between Rab7 and protrudin in mock-silenced or protrudin-silenced cells expressing protrudin wt -GFP or protrudin FYVE4A -GFP exposed to either vehicle or apilimod. All experiments were repeated three independent times and colour-code matched. Shown is the mean ± SEM. Data for (B-F) and (I) were analyzed using two-way ANOVA and Tukey’s multiple comparison test; Data for (G-H) were analyzed using a one-way ANOVA and Tukey’s multiple comparison test. P values are shown.

Article Snippet: Cells were then subjected to mouse monoclonal (DM1A) antibody against Ill-tubulin (1:100; #T9026; Sigma-Aldrich), rabbit monoclonal (D9F52) antibody against Rab7 (1:100; #9367), mouse monoclonal (E9O7E) antibody against Rab7 (1:100; #95746), mouse monoclonal (D4O1S) antibody against LAMP1 (1:100; #15665) and rabbit monoclonal (D2D11) antibody against LAMP1 (1:200; #9091; all from Cell Signaling Technology).

Techniques: Transfection, Control, Labeling, Fluorescence, Expressing, Comparison

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A-D) Confocal images of HeLa cells treated with 0.02% vehicle (DMSO), 240 nM apilimod, or 1 µM YM-201636 for 2 h followed by Proximity Ligation Assay between protrudin and VapA (A), or Rab7 and VapA (B), or Rab7 and protrudin (C), or Rab7 and Kinesin-1 (KIF5B; D). Images were generated by maximum projection of z-stacks of the PLA signal (magenta); DAPI-stained nucleus is shown in gray. Negative controls where one of the primary antibodies was missing are shown. Scale bar = 15 µm. (E-H) Quantification of the number of PLA dots per cell for each protein pair tested in A-D, as indicated in image. All experiments were repeated four independent times with matching independent experiments colour coded. Shown is the mean ± SEM. Data were analyzed using an Ordinary one-way ANOVA and Dunnett’s multiple comparisons test and p values are shown.

Article Snippet: Cells were co-incubated with one mouse and one rabbit antibody from the following; rabbit monoclonal (D9F52) antibody against Rab7 (1:100; #9367; Cell Signaling Technology), mouse monoclonal (E9O7E) antibody against Rab7 (1:100; #95746; Cell Signaling Technology), mouse monoclonal (4C12) antibody against VAPA (1:100; #293278; Santa Cruz Biotechnology), rabbit polyclonal antibody against ZFYVE27 (1:100; 12680-1-AP; Proteintech), rabbit monoclonal (E3H4J) antibody against KIF5B (1:250; #47610; Cell Signaling Technology), followed by incubation with PLA probes.

Techniques: Proximity Ligation Assay, Generated, Staining

Journal: bioRxiv

Article Title: PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

doi: 10.1101/2025.07.15.664974

Figure Lengend Snippet: (A) Confocal images of HeLa cells co-transfected with non-targeting siRNA control or siRNA oligonucleotides against protrudin, and protrudin wt -GFP or protrudin FYVE4A -GFP (gray). Cells were labeled with Dextran, Alexa 546 (magenta) and then exposed to either 0.02% vehicle (DMSO) or 240 nM apilimod for 2 h. Scale bar: full size = 20 µm, zoom insert = 5 µm. (B-D) Quantitative analysis of ER morphology based on protrudin wt -GFP or protrudin FYVE4A -GFP fluorescence in conditions shown in A: number of protrudin branches per cell (B), junctions per cell (C), and average branch length (D). (E, F) Quantification of number of PLA dots between Rab7 and protrudin (E) and Rab7 and VapA (F) in control silenced and protrudin-silenced cells with and without apilimod. (G, H) Quantification of PLA dots between Rab7 and protrudin (G) and Rab7-VapA (H) in cells treated with vehicle, apilimod, or Vps34-IN to abate PtdIns(3)P levels. (I) Quantification of PLA dots between Rab7 and protrudin in mock-silenced or protrudin-silenced cells expressing protrudin wt -GFP or protrudin FYVE4A -GFP exposed to either vehicle or apilimod. All experiments were repeated three independent times and colour-code matched. Shown is the mean ± SEM. Data for (B-F) and (I) were analyzed using two-way ANOVA and Tukey’s multiple comparison test; Data for (G-H) were analyzed using a one-way ANOVA and Tukey’s multiple comparison test. P values are shown.

Article Snippet: Cells were co-incubated with one mouse and one rabbit antibody from the following; rabbit monoclonal (D9F52) antibody against Rab7 (1:100; #9367; Cell Signaling Technology), mouse monoclonal (E9O7E) antibody against Rab7 (1:100; #95746; Cell Signaling Technology), mouse monoclonal (4C12) antibody against VAPA (1:100; #293278; Santa Cruz Biotechnology), rabbit polyclonal antibody against ZFYVE27 (1:100; 12680-1-AP; Proteintech), rabbit monoclonal (E3H4J) antibody against KIF5B (1:250; #47610; Cell Signaling Technology), followed by incubation with PLA probes.

Techniques: Transfection, Control, Labeling, Fluorescence, Expressing, Comparison