Journal: The Journal of Neuroscience

Article Title: ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells

doi: 10.1523/JNEUROSCI.2658-17.2017

Figure Lengend Snippet: Hair-cell localization of proteins involved in apical membrane trafficking. Displayed for each protein (in type I or type II hair cells) are an x-z reslice showing the protein's apical-basal profile, as well as x-y slices at apical, prenuclear, and nuclear levels. A–F, Type I hair cells. G–L, Type II hair cells. A, G: ARF6. B, H: CLTC. C, I: EEA1. D, J: LAMP1. E, K: RAB11. F, L: RAB5. Panel full widths: 18 μm.

Article Snippet: For ARF6 and other membrane markers, the following dilutions were used: 1:100 anti-ARF6, 1:250-activated ARF6 (NewEast Biosciences, 26918; RRID: AB_2629397 ); 1:250 anti-clathrin heavy chain (X22, MA1-065, ThermoFisher Scientific; RRID: AB_2083179 ), 1:250 anti-EEA1 (3288, Cell Signaling Technology; RRID: AB_2096811 ), 1:250 anti-Rab11 (5589, Cell Signaling Technology; RRID: AB_10693925 ), 1:250 anti-Rab5 (3547, Cell Signaling Technology; RRID: AB_2300649 ), and 1:250 anti-LAMP1 (H4A3, Developmental Studies Hybridoma Bank; RRID: AB_2296838 ).

Techniques:

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion does not affect Rab5 recruitment to the endosomes and ligand-bound EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab5 and EEA1 (A), Rab5 and CD63 (B), and Rab5 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Right (A–C): single Z-plane of images. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab5 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab5 staining quantified in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Scale bars: 10 μm. p values were not statistically significant. Zoomed images correspond to the dashed inset boxes of the indicated images. Note: (B) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (B) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (B) and (C) is intentional.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab5 antibody , Proteintech , 20228-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: EGFR activated by HB-EGF accumulation in Rab5-positive endosomes is specific to HD-PTP depletion. Control HeLa cells or cells depleted of HD-PTP or Alix were incubated with fluorescently labelled EGFR, stimulated with HB-EGF, chased for up to 180 min and immunostained for Rab5. Arrows indicate colocalised Rab5 and EGFR puncta; arrowheads indicate undegraded EGFR not colocalised with Rab5-positive endosomes. Asterisks indicate nuclei from binucleate cells. Representative data from three independent experiments. The percentages indicate the proportion of EGFR-containing structures that also labelled for Rab5. Scale bar: 10 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Incubation

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: The effect of HD-PTP depletion on endosomal clustering is not secondary to ESCRT depletion. (A) Endosomal clusters induced by HD-PTP depletion is not an off-target effect. HeLa cells were depleted with siRNA and rescued with RNAi-resistant HA–HD-PTP as indicated and immunostained for Rab5 and EEA1. Graph shows quantification of the approximate area of endosomal structures. Values represent mean±s.d. ( n =10). *** P <0.0001 (one-way ANOVA with Bonferroni's test for multiple comparisons). (B) Validation of siRNA silencing of VPS4B. Values represent mean±s.d. ( n =4). *** P <0.0001 (unpaired two-tailed Student's t -test). (C) Aberrant ESCRT-III accumulation caused by HD-PTP depletion is not responsible for clustering of Rab5 endosomes. HeLa cells were depleted with siRNA as indicated and immunostained for Rab5 and CHMP4B. Representative data from three independent experiments. Scale bars: 20 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Two Tailed Test

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: Depletion of HD-PTP enhances Rab5 activity on endosomes. (A) HeLa cells were depleted with siRNA as indicated and immunostained for EEA1. Histogram shows the percentage of cells that displayed a normal distribution of EEA1 (mean±s.d. from at least three independent experiments, 100–300 cells counted per experiment, total number of cells indicated above each histogram bar). *** P <0.0001; ns, not significant (one-way ANOVA with Bonferroni's test for multiple comparisons). RBPT5, Rabaptin-5. (B) As A, but immunostained for Rab5. (C) HeLa cells were depleted with siRNA as indicated and transfected with NowGFP–Rab5a and mRuby–EEA1.sh-R5BD. The fluorescence lifetime of NowGFP–Rab5a was measured. Panels show the scale and distribution of fluorescence lifetime in representative cells (lookup chart key on right, black/blue=0 ns, red=7000 ns). Arrowheads indicate pockets of decreased fluorescence lifetime. Graph shows quantification of fluorescence lifetimes over whole cell areas. Values represent mean±s.d. ( n =8–18 cells from three independent experiments, total number of cells indicated above each histogram bar). *** P <0.0001, ns, not significant (one-way ANOVA with Bonferroni's test for multiple comparisons). Scale bars: 20 µm.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Activity Assay, Transfection, Fluorescence

Journal: Journal of Cell Science

Article Title: His domain protein tyrosine phosphatase and Rabaptin-5 couple endo-lysosomal sorting of EGFR with endosomal maturation

doi: 10.1242/jcs.259192

Figure Lengend Snippet: Model of the coupling of endosomal sorting and maturation. (i) HD-PTP initiates ESCRT-dependent MVB sorting of cargo by binding STAM2/ESCRT-0 while Rabex-5–Rabaptin-5 engages ubiquitylated cargo and is active to maintain high GTP-Rab5. (ii,iii) As cargo sorting proceeds, HD-PTP releases STAM2/ESCRT-0 and then binds CHMP4/ESCRT-III, while cargo is deubiquitylated. (iv) Cargo enters ILVs as CHMP4B/ESCRT-III is released. (v) HD-PTP, now free from ESCRTs, bind to Rabaptin-5. Rabex-5–Rabaptin-5 is inhibited, thus lowering GTP-Rab5 and promoting Rab conversion.

Article Snippet: Rabbit: anti-CHMP4B (Proteintech; Cat. 13683-1-AP; batch 00019545; 1:250 for IF); anti-HD-PTP (Proteintech; Cat. 10472-1-AP; batch 00018154; 1:5000 for WB; 1:50 for IF) ( ); anti-UBAP1 (Proteintech; Cat. 12385-1-AP; batch 00014891) ( ); anti-EEA1 (Cell Signaling Technologies; C45B10; Cat. 3288S; batch 8; 1:400 for IF); anti-Rabex-5 (Thermo Scientific - Invitrogen; Cat. PA5-21117; batch RF2221849; 1:2500 for WB); anti-Rabaptin-5 (Proteintech; Cat. 14350-1-AP; batch 00005401; 1:5000 for WB); anti-Rab5 clone C8B1 (Cell Signaling Technologies; Cat. 3547S; batch 7; 1:500 for IF; 1:2500 for WB); anti-GAPVD1 (Abcam; Cat. ab86033; batch GR265707-8; 1:5000 for WB); anti-VPS4B (Proteintech; Cat. 17673-1-AP; batch 00009255; 1:2500 for WB); anti-exportin-1 (Bethyl Laboratories; Cat. A300-469A; batch 2).

Techniques: Binding Assay