gfp rab5  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is recruited to lipid droplets (LDs) in a GTPase-dependent manner in Hep3B cells . Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing ( A ) WT Rab5 (GFP-Rab5 WT), ( B ) constitutively active mutant (GFP-Rab5 Q79L), and ( C ) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red ) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green ) marks Rab5 localization. Inlays depict Rab5 localization around LD borders, indicated further by yellow arrows . D , quantification of Rab5–LD colocalization by the ratio of LD:cytoplasm GFP-Rab5 intensity. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The GFP-Rab5 was a gift from Marci Scidmore (Addgene plasmid #49888).

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, 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: GFP-Rab5 plasmid , Addgene , 56417.

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: 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: GFP-Rab5 DN plasmid , Addgene , 28045.

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

Journal: bioRxiv

Article Title: RAB5 NUCLEOTIDE BINDING PROMOTES β-OXIDATION TO FUEL HEPATOCELLULAR CARCINOMA CELL PROLIFERATION

doi: 10.1101/2025.08.20.670915

Figure Lengend Snippet: Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing (A) wild-type Rab5 (GFP-Rab5 WT), (B) constitutively active mutant (GFP-Rab5 Q79L), and (C) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green) marks Rab5 localization. Each panel contains two high-magnification insets (i and ii): inset i and ii zooms in on Rab5 puncta at LD borders, while below each of this insets shows the grayscale GFP channel alone, highlighting Rab5 signal independent of ORO. In Panel A, yellow arrows indicate discrete punctate Rab5 structures in close proximity to ORO-stained LDs, representing moderate LD association by wild-type Rab5. In Panel B, yellow arrows highlight continuous GFP-Rab5 rings surrounding smaller LDs, showing enhanced recruitment of the GTP-locked Rab5 Q79L to LD surfaces. In Panel C, yellow arrows mark areas where LDs lack adjacent Rab5 signal, and the GFP-Rab5 S34N mutant remains diffusely distributed throughout the cytoplasm, confirming a failure in membrane targeting due to its GDP-bound inactive state. (D) Quantification of Rab5-LD co-localization. The LD/Cytoplasm Intensity Ratio was calculated for each mutant by dividing the average GFP-Rab5 intensity at LD surfaces by the cytoplasmic GFP-Rab5 intensity. Bars represent mean ± SD from 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. **p<0.01 and ***p<0.001. Scale bars: main images, 10 µm; insets, 2 µm.

Article Snippet: The constitutively active GFP-Rab5 (Q79L) and dominant-negative GFP-Rab5 (S34N) constructs were gifted from Dr. Sergio Grinstein (Addgene plasmids #35140 and #35141, respectively).

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence, Membrane

Journal: bioRxiv

Article Title: MEG3 Enhances Survival of Developing Human Neurons with CLCN4 -Linked Autophagy Impairment

doi: 10.1101/2025.07.16.665078

Figure Lengend Snippet: (a) Representative images of day 6 neurons after Lysotracker treatment. Lysotracker is shown in magenta, and DAPI in blue. Scale bar = 20 μm. (b) Quantification of Lysotracker signal in day 6 neurons. The relative Lysotracker intensity of CLCN4 -variant neurons compared to the WT was presented. Each dot represents one biological replicate. (n=3-4, mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test * p =0.039 for variant A; *** p =0.0007 for variant B; ** p =0.0092 for variant C) The magenta dashed line indicates the value for WT. (c) Relative acidity in acidic subcellular organelles. Each dot represents one biological replicate. (n=3, mean ± SEM, unpaired Student’s t-test; p =0.2894) (d-g) Dynamics of endo-lysosomal vesicles. RAB5, RAB7, RAB11 and LAMP1 indicates early endosome, late endosome, recycling endosome and lysosome markers, respectively. (RAB5, n=6; RAB7, n=3; RAB11, n=4; and LAMP1, n=3 or 4; mean ± SEM, unpaired Student’s t-test) (d) Representative images of a neuron with GFP-labeled vesicular organelles. TUJ1 and DAPI are shown in red and blue, respectively. Scale bar = 10 μm. (e) Relative GFP intensity expressed in vesicular organelles (* p =0.0265 for RAB5, * p =0.0158 for RAB7, * p =0.0475 for RAB11, and ** p =0.0067 for LAMP1) (f) Number of GFP-expressing vesicles in neurons. (* p =0.0186 for RAB5, * *p =0.0066 for RAB7, and p =0.9031 for LAMP1) (g) Average distance of GFP-expressing vesicles from the center of nucleus, representing the mean linear distance of puncta from the center of the nucleus in each cell. (** p =0.0011 for RAB5, * p =0.0463 for RAB7, and * p =0.0103 for LAMP1) (h) Scheme of autophagic flux analysis using tandem fluorescent protein fused to LC3. (i) Representative images of day 6 neurons expressing tandem fluorescent-tagged LC3. Red: RFP, green: GFP, white: TUJ1. Scale bar = 5 μm. (j) Quantification of the GFP-/RFP+ puncta. (n=5, mean ± SEM, unpaired Student’s t-test; * p =0.0164)

Article Snippet: For specific subcellular vesicle visualization, lentiviral constructs encoding GFP-tagged RAB5 (Addgene #134858), RAB7 (Addgene #133027), RAB11 (Addgene #134860), and LAMP1 (Addgene #134868) were utilized.

Techniques: Variant Assay, Labeling, Expressing