gfp rab7 plasmid  (Addgene inc)

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: ANXA3 participated in T2DM-induced DbCM by regulating microlipophagy. (A-B) Representative images of TEM were used to observe the autophagy and microlipophagy in the hearts of mice. The red arrow represents autophagosomes, and the yellow arrow represents lipid droplets in contact with lysosomes. Bars: 1 μm (C) Relative Rab7a mRNA expression levels in heart tissues of mice analyzed by RT-qPCR, n = 4. (D-E) Relative Rab7a protein expression levels in the heart of mice analyzed by Western blot, n = 4. (F) Relative RAB7A mRNA expression levels in AC16 cells analyzed by RT-qPCR, n = 3-6. (G-H) Relative Rab7a protein expression levels in AC16 cells analyzed by Western blot, n = 3-5. The data were presented as the mean ± SD. ∗ P < 0.05 versus the db/db + OE-GFP group or the si-NC group; # P < 0.05 versus the OE-NC group.

Article Snippet: Blocked with 5% (w/v) non-fat-dried milkat room temperature for 1 h. Then the membranes were incubated with the anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:1000 [v/v]), the anti-YY1 antibody (Proteintech, 22156-1-AP; 1:1000 [v/v]) the anti-PLIN2 antibody (Proteintech, 15294-1-AP; 1:1000 [v/v]), the anti-SQSTM1/p62 antibody (Cell Signaling Technology, 23214; 1:1000 [v/v]), the anti-LC3 antibody (Cell Signaling Technology, 12741; 1:1000 [v/v]), the anti-Rab7 antibody (Proteintech, 55469-1-AP; 1:1000 [v/v])or the rabbit anti-α-Tubulin antibody (ABclonal, AC031; 1:1000 [v/v])overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

doi: 10.64898/2026.03.14.711762

Figure Lengend Snippet: (A) H522 TMEM106B KO cell derivatives stably transduced with an empty vector (EV), and HA-tagged full length TMEM106B (FL), TMEM106BΔCTD, TMEM106BΔNTD were subjected to immunofluorescence microscopy. Detection of TMEM106B variants (anti-HA, green), Rab7a (lysosome marker, in red), wheat germ agglutinin (membrane, cyan), and cellular nuclei (DAPI, blue). Scale bar=10μM. (B, C) BHK-21 cells stably expressing CD71-TMEM106B-CTD-HA (CD71-CTD-HA) chimeric protein were subjected to western blotting ( B ) or surface staining and flow cytometry ( C ) using an anti-HA antibody. (D, E) BHK21-CD71-TMEM106B-CTD-HA cells were inoculated with SARS-CoV-2 mNG S E484D at a MOI of 1. Infected cells were subjected to FACS to enumerate mNG positive cells at 48 h post-infection (D) or RT-qPCR analysis to analyze cell-associated SARS-CoV-2 RNA at 48 h post infection (E). ( F ) BHK21-CD71-TMEM106B-CTD cells were infected as in (D, E) but in the presence of 50 mM ammonium chloride as in . Cell-associated SARS-CoV-2 RNA levels were assessed by RT-qPCR at 48 h post infection. ( G, H ) SARS-CoV-2 S E484D (MOI=2 equivalent) was pre-incubated with the indicated concentrations of Fc-TMEM106B-LD in SF-DMEM at 37°C for 30 min. Fc decoy-virus mixture was added on H522 (G) and H661 (H) cells and incubated at 37°C for 2h. Virus inoculum was removed and cells were replenished with complete culture media. Cell-associated viral RNA was analyzed by RT-qPCR at 72 h post infection. Data in E-H show the average of three independent replicates with error bars displaying the SEM. ** P < 0.01; *** P < 0.001; *** P < 0.0001 by two-tailed unpaired t -test or one-way ANOVA with Dunnett’s correction for multiple comparisons.

Article Snippet: After permeabilization, cells were incubated in 1% bovine serum albumin (BSA) and 10% FBS in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 h. Samples were then incubated in primary mouse monoclonal anti-HA (Biolegend, clone 16B12, 1:500) and rabbit polyclonal anti Rab7a (Novus Biologicals #NBP1-87174, 1:500) antibodies at 4 °C overnight.

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Immunofluorescence, Microscopy, Marker, Membrane, Expressing, Western Blot, Staining, Flow Cytometry, Infection, Quantitative RT-PCR, Incubation, Virus, Two Tailed Test

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 Rab7 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 Rab7 and EEA1 (A), Rab7 and CD63 (B), and Rab7 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Middle (A and B) and right (C): single Z-plane of images. Right (A): co-localization (yellow) was quantified by determining the percentages of EEA1-positive (red) puncta containing Rab7 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells from 60 min time point of one representative experiment. Right (B): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab7 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells at 60 min time point of one representative experiment. The object-based co-localization macro tool FIJI was employed. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab7 (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 Rab7 staining quantified 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, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test. Note: (A) 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 (A) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (A) and (C) is intentional.

Article Snippet: Rab7 antibody , Proteintech , 55469-1-AP.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence, 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: Rab7 antibody , Proteintech , 55469-1-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: Rab7 antibody , Proteintech , 55469-1-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: Rab7 antibody , Proteintech , 55469-1-AP.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, 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-Rab7 plasmid , Addgene , 61803.

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