rab7  (Cell Signaling Technology Inc)

Journal: Advanced Science

Article Title: Early‐Life Manganese Overexposure Activates Microglial Phagocytosis through the YY1/TREM2 Axis Leading to Hippocampal Synaptic Remodeling in Mice

doi: 10.1002/advs.78036

Figure Lengend Snippet: YY1 transcriptionally regulates TREM2 and activation of the microglial phagolysosomal pathway. (A) Representative immunofluorescence and 3D reconstruction images showing microglia phagocytosis of latex beads under different Mn exposure concentrations. Scale bar, 10 µm, magnification 3D model scale bar, 5 µm. (B,C) Quantification of fold changes in microglial phagocytic volume relative to the Control group and the proportion of phagocytic microglia within the microscopic field. (D–F) Representative fluorescence images and quantitative analysis of LysoTracker‐Red and PH‐LysoSensor Yellow/Blue signals in microglia under different Mn exposure. Scale bar, 20 µm. (G) Representative fluorescence images showing activation of the phagocytic (RAB7, green) lysosomal (LAMP1, red) pathway in microglia under different Mn exposure, with Pearson's colocalization coefficients. Scale bar, 20 µm, magnification scale bar, 10 µm. (H,I) Representative fluorescence images and quantitative analysis of nuclear YY1 (green) expression in IBA1 + (red) microglia under different Mn exposure concentrations. Scale bar, 20 µm, magnification scale bar, 10 µm. (J) Heatmap and quantitative analysis of signal intensity for each YY1 CUT&Tag sequencing peak in Control and Mn (25 µ m ) groups. (K) Distribution of YY1 CUT&Tag sequencing peaks relative to transcription start sites in control and Mn (25 µ m ) groups. (L,M) GO and KEGG enrichment analyses of DEGs identified from YY1 CUT&Tag sequencing. (N) CUT&Tag results showed YY1 binding sites in gene Trem2 . (O) Quantitative CUT&Tag qPCR results showing the relative enrichment of YY1 at Trem2 and phagolysosomal pathway ( Rab7 and Lamp1 ) with various Mn exposure, relative to Control. (P–T) Western blot analysis of YY1, TREM2, and phagolysosomal pathway (LAMP1 and RAB7) in microglia under different Mn exposure concentrations, protein expression levels normalized to β‐actin and relative to Control. Data were expressed as mean ± SD. Sample sizes n = 3 independent experiments for Western blot analysis, and n = 4 independent experiments for other assays. One‐way ANOVA followed by Fisher's LSD post hoc test. * p < 0.05, ** p < 0.01 versus Control group. # p < 0.05, # # p < 0.01 versus Mn (25 µ m ) group. && p < 0.01 versus lgG group. See also Figures and .

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β‐actin (Proteintech, China), Histone H3 (CST, USA), TREM2 (Abcam, USA), LAMP1 (CST, USA), RAB7 (CST, USA), YY1 (CST, USA), HIF1α (CST, USA), SMURF2 (Invitrogen, USA), and anti‐Ubiquitin (Invitrogen, USA).

Techniques: Activation Assay, Immunofluorescence, Control, Fluorescence, Analysis, Expressing, Sequencing, Binding Assay, Western Blot

Journal: Advanced Science

Article Title: Early‐Life Manganese Overexposure Activates Microglial Phagocytosis through the YY1/TREM2 Axis Leading to Hippocampal Synaptic Remodeling in Mice

doi: 10.1002/advs.78036

Figure Lengend Snippet: Suppression of Yy1 alleviates the excessive of TREM2‐mediated phagocytosis and activation of the phagolysosomal pathway in Mn‐exposed microglia. (A,B) Representative immunofluorescence staining and quantification of YY1 nuclear localization in IBA1 + (red) microglia across different treatment groups. Scale bar, 20 µm, magnification scale bar,10 µm. (C,D) Western blot showing YY1 expression in nuclear and quantification. (E–G) Representative images and 3D reconstructions of latex bead phagocytosis in microglia from various treatment group, with quantitative analysis of phagocytic capacity (relative to Control) and phagocytic microglia rate (%) within the microscopic field. Scale bar, 20 µm. (H) mRNA expression of Trem2 and phagolysosomal pathway–related genes ( Rab7 and Lamp1 ) in different treatment groups determined by RT‐qPCR. (I–L) Western blot analysis showing relative protein expression levels of TREM2 and phagolysosomal pathway (RAB7 and LAMP1) in microglia under different treatments. (M–O) Representative 3D reconstruction images of IBA1 + microglia within the microscopic field illustrating engulfment of PSD95 and VGAT, along with quantification of phagocytic volume. Scale bar, 10 µm. (P,Q) Representative fluorescence Z‐stack projection images reconstructed using Imaris 3D software, showing colocalization of VGLUT1 (green) and PSD95 (red), and VGAT (red) and Gephyrin (green), along with Pearson's correlation coefficient analysis and quantification of ES/IS counts relative to Control. Scale bar, 50 µm. mRNA expressions were normalized to Gapdh and relative to Control, proteins expressions were normalized to Histone H3 or β‐actin and relative to Control. Data were expressed as mean ± SD. For Western blot analysis, n = 3 independent experiments; and for ex vivo brain slice and other experiments, n = 4 independent samples per group. Data were analyzed by two‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01 versus Control group. # p < 0.05, # # p < 0.01 versus Mn group. See also Figures and .

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β‐actin (Proteintech, China), Histone H3 (CST, USA), TREM2 (Abcam, USA), LAMP1 (CST, USA), RAB7 (CST, USA), YY1 (CST, USA), HIF1α (CST, USA), SMURF2 (Invitrogen, USA), and anti‐Ubiquitin (Invitrogen, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Analysis, Control, Quantitative RT-PCR, Fluorescence, Software, Ex Vivo, Slice Preparation

Journal: Advanced Science

Article Title: Early‐Life Manganese Overexposure Activates Microglial Phagocytosis through the YY1/TREM2 Axis Leading to Hippocampal Synaptic Remodeling in Mice

doi: 10.1002/advs.78036

Figure Lengend Snippet: HIF1α regulation and dysfunction of SMURF2‐mediated ubiquitin degradation are responsible for YY1 upregulation. (A) Predicted HIF1α binding sites in the Yy1 promoter region from the JASPAR database. (B) Co‐expression analysis of Hif1a and Yy1 in the hippocampus by GEPIA database. (C) ChIP assay showing the binding between HIF1α and the Yy1 promoter. (D,E) Representative immunofluorescence images and quantitative analysis showing the nuclear expression of YY1 under different treatments. Scale bar, 20 µm, magnification scale bar, 10 µm. (F,G) Western blot analysis of nuclear YY1 protein levels in microglia from different treatment groups. (H) RT‐qPCR analysis of Yy1 , Trem2 , and phagolysosomal pathway‐related genes ( Rab7 and Lamp1 ) under various treatments. (I–M) Western blot and quantification of YY1, TREM2, and phagolysosome (RAB7 and LAMP1) proteins under different treatments. (N,O) Western blot showing YY1 protein degradation dynamics following CHX treatment at different time points under Mn exposure. (P) Immunoprecipitation analysis of YY1 ubiquitination levels in microglia. (Q) Identification of the E3 ubiquitin ligase SMURF2 targeting YY1 by the UbiBrowser 2.0 database. (R) Relative mRNA expression level of Smurf2 in Mn treatment assessed by RT‐qPCR. (S,T) Western blot and quantification of SMURF2 relative protein expression level under Mn treatment. (U) Molecular docking prediction of potential Mn 2 + binding sites within SMURF2. (V,W) Co‐immunoprecipitation assays revealing the interaction strength between SMURF2 and YY1 under different treatments. (X) Analysis of YY1 ubiquitination levels after immunoprecipitation across different experimental groups. mRNA expressions were normalized to Gapdh and relative to Control, proteins expressions were normalized to Histone H3 or β‐actin and relative to Control. Data were expressed as mean ± SD. For in vitro experiments, n = 3 independent experiments for Western blot analysis, and n = 4 independent experiments for all other assays. Data were analyzed by two‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01 versus Control group. ## p < 0.01 versus Mn (25 µ m ) group. See also Figure .

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β‐actin (Proteintech, China), Histone H3 (CST, USA), TREM2 (Abcam, USA), LAMP1 (CST, USA), RAB7 (CST, USA), YY1 (CST, USA), HIF1α (CST, USA), SMURF2 (Invitrogen, USA), and anti‐Ubiquitin (Invitrogen, USA).

Techniques: Ubiquitin Proteomics, Binding Assay, Expressing, Analysis, Immunofluorescence, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Docking Assay, Co-Immunoprecipitation Assay, Control, In Vitro

Journal: Frontiers in Cardiovascular Medicine

Article Title: hTERT associates with Rab7A to enhance autophagy and rescue oxLDL induced endothelial dysfunction

doi: 10.3389/fcvm.2026.1882233

Figure Lengend Snippet: hTERT associates with Rab7A to regulate autophagy. (A) Immunofluorescence images showing expression of hTERT and Rab7A under 40 µg/mL oxLDL treatment of HUVEC cells (B) Enlarged images showing colocalization of hTERT and Rab7A upon oxLDL treatment indicated by arrows. (C) Graph indicates quantification of colocalization using Pearson's correlation plugin in Fiji software. (D,E) Co-immunoprecipitation experiments showing association of hTERT with autophagy related proteins at basal (D) oxLDL treated condition (E,F) Densitometric analysis of hTERT and Rab7A shown in western blots in (E) (G) Enlarged images of DiI-oxLDL in HUVEC cells under Rab7DN, hTERT overexpression (hTERT Oe) and hTERT knockdown (hTERT Kd) conditions. (H) Quantification of DiI-oxLDL fluorescence intensity under different conditions (I) Schema showing possible association step of hTERT in autophagy regulation (Created using BioRender.com). Scale bar=100 µm (A) , 50 µm (B,F) , Data are representative of n = 3 biological replicates, Data are presented as Mean ± SEM, Unpaired two-tailed t-test, * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: Rab7A based experiments were performed using Rab7DN plasmid [A kind gift from Richard Pagano (Addgene plasmid # 12660)] by transfecting 500 ng of plasmid into cells in a 12 well plate.

Techniques: Immunofluorescence, Expressing, Software, Co-Immunoprecipitation Assay, Analysis, Western Blot, Over Expression, Knockdown, Fluorescence, Two Tailed Test

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 interacts with the lysosomal protein Rab7 to contribute to macrophage inflammation. (A) Representative top five proteins potentially binding to SH3GLB1 in mitochondria identified by mass spectrometry analysis. (B) Representative western blots of Rab7 protein levels in BMDMs treated with LPS or sea water. (C) Co-IP assay showing the interaction of SH3GLB1 with Rab7. (D) Representative images of immunofluorescence staining for SH3GLB1 (green) and Rab7 (red) in BMDMs. Scale bar, 20 µ m. (E) Representative molecular docking analysis of SH3GLB1 and Rab7. (F) Western blots of recombinant Rab7 binding to recombinant SH3GLB1. Representative ELISA results showing the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (G and H) LPS (n=3) or (I and J) sea water (n=3). The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; BMDMs, bone marrow-derived macrophages; LPS, lipopolysaccharide; Co-IP, coimmunoprecipitation.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Binding Assay, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Macrophage SH3 domain-containing GRB2-like protein B1 is required for Rab7-mediated mitophagy in inflammation during sea water drowning acute lung injury

doi: 10.3892/ijmm.2026.5809

Figure Lengend Snippet: SH3GLB1 facilitated adverse Rab7-mediated mitophagy. (A) Representative images of immunofluorescence staining for SH3GLB1 (green) and mitochondria (red) or lysosomes (red) in BMDMs. Scale bar, 20 µ m. Representative (B and F) western blot and (C-E and G-I) quantification of mitophagy-related protein levels in BMDMs treated with CCCP. (J) mKeima expressed in BMDMs was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar, 20 µ m); (K) the 561/488 nm ratio of mKeima was statistically quantified (n=3). (L) Representative flow cytometry analysis of mKeima in BMDMs. The data are presented as the means ± SEMs; P<0.05 was considered to indicate statistical significance. SH3GLB1, SH3 domain-containing GRB2-like protein B1; BMDMs, bone marrow-derived macrophages; CCCP, carbonyl cyanide-chlorophenylhydrazone.

Article Snippet: Primary antibodies: GAPDH (1:1,000; cat. no. ab8245; Abcam), HSP90 (1:1,000; cat. no. 13171-1-AP, Proteintech Group, Inc.), SH3GLB1 (1:1,000; cat. no. sc-374146, Santa Cruz Biotechnology, Inc.), LC3 (1:1,000; cat. no. 12741T; CST Biological Reagents Co., Ltd.), P62 (1:1,000; cat. no. ab109012, Abcam), Rab7 (1:1,000; cat. no. A12308, ABclonal) and Parkin (1:1,000; cat. no. ab77924, Abcam).

Techniques: Immunofluorescence, Staining, Western Blot, Confocal Microscopy, Flow Cytometry, Derivative Assay