sh3glb1  (Santa Cruz Biotechnology)

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: The mitochondrial protein SH3GLB1 was highly expressed in alveolar macrophages in drowning-ALI. (A) Representative TEM images of AM mitochondria in the lung (the red arrow indicates injured and swollen mitochondria; scale bars, 2 µ m/500 nm). (B) Representative analysis of single-cell sequencing data and identification of the top 15 mitochondrial proteins. (C) Representative macrophage subcluster-specific expression patterns of SH3GLB1 between the sham and sea water groups. (D) UMAP analysis and (E) percentages of high and low SH3GLB1 expression in AMs treated with sham or sea water; colored unsupervised clustering results with cell type annotations are shown on the right. (F) Relative SH3GLB1 mRNA levels in alveolar macrophages from shamor sea water-induced ALI model mice (n=7). (G) Representative western blots of SH3GLB1 protein levels in shamor sea water-induced ALI model mice. 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; ALI, acute lung injury; UMAP, uniform manifold approximation and projection; TEM, transmission electron microscopy; AMs, alveolar macrophages.

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: Single Cell, Sequencing, Expressing, Western Blot, Transmission Assay, Electron Microscopy

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 deficiency protects against inflammation in ALI. (A) Gene expression of SH3GLB1 high AMs in cohort 1 (n=3) represented in a volcano plot, with the fold change (log2) expressed as the level in sham versus sea water-ALI subjects on the x-axis and the P-value on the y-axis (log10). Significantly (P<0.05) regulated genes are marked in colors: red indicates upregulated genes and blue indicates downregulated genes in ALI. (B) Representative GO analysis of SH3GLB1 high AMs. (C) Representative KEGG analysis of SH3GLB1 high AMs. (D) Representative hematoxylin and eosin staining of lung tissues (scale bars, 200 µ m). (E) Representative severity scores of lung injury (n=3). Representative ELISA results showing the levels of inflammatory factors, including (F) TNFα, (G) IL-6 and (H) IL-1β, in the BALF (n=6). (I-K) Representative ELISA analysis of the levels of inflammatory factors, including (I) IL-1β, (J) IL-6 and (K) TNFα, in the serum (n=6). (L) Representative western blots of SH3GLB1 protein levels in NC or SH3GLB1-KD BMDMs. (M) Relative SH3GLB1 mRNA levels in (NC or SH3GLB1-KD BMDMs. n=6/group. Representative ELISA analysis of the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (N) LPS or (O) seawater, (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; AMs, alveolar macrophages, ALI, acute lung injury; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BALF, bronchoalveolar lavage fluid; BMDMs, bone marrow-derived macrophages; KD, knockdown; WT, wild-type; KO, knockout; NC, negative control; LPS, lipopolysaccharide.

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: Gene Expression, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Knockdown, Knock-Out, Negative Control

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: Reduced SH3GLB1 ameliorates LPS-induced mitochondrial dysfunction in macrophages. (A) Representative TEM images of macrophage mitochondria in the lung (scale bars, 2 µ m/500 nm). Representative (B) confocal microscopy and (C) flow cytometry analysis of the mitochondrial membrane potential by JC-1 staining in BMDMs. A high level of green fluorescence (x-axis) represents a reduced mitochondrial membrane potential (ΔΨm) and a high level of red fluorescence (y-axis) represents a normal ΔΨm. The red fluorescence rate was analyzed (n=3). (D) Representative confocal microscopy images of mitochondrial ROS production (MitoSOX fluorescence; red) in NC or SH3GLB1-KD BMDMs under normal or LPS conditions (scale bar, 50 µ m). (E) Analysis of the OCR of mitochondrial respiratory capacity, including basal respiration, ATP production, maximal respiration and spare respiration, in NC or SH3GLB1-KD BMDMs under normal or LPS conditions. 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; LPS, lipopolysaccharide; TEM, transmission electron microscopy; BMDMs, bone marrow-derived macrophages; KD, knockdown; OCR, oxygen consumption rate; NC, negative control.

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: Confocal Microscopy, Flow Cytometry, Membrane, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Derivative Assay, Knockdown, Negative Control

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: Restoration of SH3GLB1 expression provokes ALI. (A) Representative hematoxylin and eosin staining of lung tissues (scale bars, 100 µ m). (B) Representative severity scores of lung injury (n=3). Representative ELISA results showing the levels of inflammatory factors, including (C) TNFα, (D) IL-1β and (E) IL-6, in the BALF (n=6). Representative ELISA analysis of the levels of inflammatory factors, including (F) IL-1β, (G) IL-6 and (H) TNFα, in the serum (n=6). (I) Western blots of SH3GLB1 expression. (J) Relative SH3GLB1 mRNA levels in vectoror SH3GLB1-overexpressing BMDMs. n=6/group. Representative ELISA analysis of the levels of inflammatory factors, including IL-1β, IL-6 and TNFα, in the supernatants of cells treated with (K) LPS or (L) 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; ALI, acute lung injury; BMDMs, bone marrow-derived macrophages; WT, wild-type; OV; overexpression.

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: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Over Expression

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 overexpression provoked mitochondrial dysfunction in macrophages. (A) Representative TEM images of macrophage mitochondria in the lung (scale bars, 2 µ m/500 nm). Representative (B) confocal microscopy and (C) flow cytometry analysis of the mitochondrial membrane potential by JC-1 staining in BMDMs (n=3). (D) Representative confocal microscopy images of mitochondrial ROS production in vectoror SH3GLB1-OV BMDMs under normal or LPS conditions (scale bar, 50 µ m). (E) Analysis of the OCR of mitochondrial respiratory capacity, including basal respiration, ATP production, maximal respiration and spare respiration, in vectoror SH3GLB1-overexpressing BMDMs under normal or LPS conditions. 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; TEM, transmission electron microscopy; OCR, oxygen consumption rate; BMDMs, bone marrow-derived macrophages.

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: Over Expression, Confocal Microscopy, Flow Cytometry, Membrane, Staining, Transmission Assay, Electron Microscopy, 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 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

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. HPA, Human Protein Atlas

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Expressing, Derivative Assay, Gene Expression

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: SH3GLB1 in HPA - pathology. ( A ) A statistical analysis was conducted on the immunohistochemical staining results of the SH3GLB1 gene across various tumor types. ( B ) Immunohistochemical sections revealed distinct differences in the protein expression levels of SH3GLB1 among different tissues. The intensity and distribution patterns observed in these immunohistochemical sections reflect both the expression level and subcellular localization of the target protein within cells

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: SH3GLB1 demonstrates both diagnostic and prognostic value across various types of cancer. ( A ) Bar plots illustrate the AUC values used to assess the diagnostic performance of SH3GLB1 expression in tumor versus normal tissues. ( B ) A heatmap depicts the association between SH3GLB1 mRNA expression levels and diverse survival outcomes in pan-cancer analysis. ( C – F ) Forest plots present the results of pan-cancer analyses evaluating the relationship between SH3GLB1 expression and overall survival (OS) ( C ), disease-free survival (DFS) ( D ), progression-free interval (PFI) ( E ), and distant metastasis-free interval (DFI) ( F )

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Diagnostic Assay, Expressing

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: Analyses of SH3GLB1 genomic alterations. ( A ) Stacked bar plots showing mutation frequencies of SH3GLB1 in pan-cancer. ( B ) Lollipop map of SH3GLB1 mutation sites in pan-cancer. ( C ) Spearman correlation between copy number variation score and SH3GLB1 mRNA expression level. ( D ) Heatmap showing mutation of SH3GLB1 and several classical carcinogenic signaling pathways in pan-cancer. ( E ) Oncoplot of the mutation distribution of SH3GLB1 in pan-cancer

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Mutagenesis, Expressing, Protein-Protein interactions

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: SH3GLB1 is implicated in multiple oncogenic pathways and exhibits a significant association with immune activity. ( A ) Bubble plots illustrating the correlation between SH3GLB1 mRNA expression levels and each hallmark gene set activity across the TCGA pan-cancer dataset. NES denotes the normalized enrichment score. ( B ) Comparative analysis of pathway activity scores between the high-expression and low-expression groups of SH3GLB1 based on data from the TCPA database. ( C ) Variations in immune subtypes among pan-cancer samples stratified by high and low SH3GLB1 mRNA expression levels. ( D ) A heatmap depicting the Pearson correlation between SH3GLB1 expression and immune-related genes across pan-cancer types

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Activity Assay, Expressing

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: SH3GLB1 in colorectal cancer: expression levels, survival analysis, and pathway enrichment. ( A ) The Beeswarm plots illustrate the correlation between the distribution of SH3GLB1 mRNA expression in normal and tumor tissues. ( B ) The forest plot presents the findings of a univariate Cox survival meta-analysis for the SH3GLB1 gene in colorectal cancer, incorporating data from the TCGA database as well as other external datasets. ( C ) The box diagram displays the results of comparing pathway activity scores between two groups of SH3GLB1 samples (Low group and High group), based on the Wilcoxon rank sum test

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Expressing, Activity Assay

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: Analysis of Immune Infiltration and Correlation of SH3GLB1 in CRC. ( A ) The heatmap illustrates disparities in microenvironmental components between SH3GLB1 high - and low - expression groups, as evaluated by seven distinct algorithms. ( B ) The heatmap displays variations in the expression of immune - stimulating genes, immunosuppressive genes, chemokines, and human leukocyte antigens between SH3GLB1 high - and low - expression groups. ( C ) The lollipop plot depicts the correlation between SH3GLB1 expression and microenvironment components as assessed by seven algorithms. ( D ) The Spearman correlation between TIP scores and SH3GLB1 gene expression levels, as well as the autocorrelation among TIP scores, is presented. ( E ) The scatter plot reveals the correlation between SH3GLB1 expression levels in immune - infiltrated cells and the abundance of various immune cells

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Expressing, Gene Expression

Journal: Biology Direct

Article Title: Comprehensive pan-cancer analysis reveals SH3GLB1 is a novel prognostic biomarker with immunomodulatory potential

doi: 10.1186/s13062-026-00728-0

Figure Lengend Snippet: ( A ) Relative expression levels of SH3GLB1 in various CRC cell lines as determined by qRT-PCR. ( B ) Western blot analysis confirming SH3GLB1 protein expression in CRC cell lines. ( C - D ) HT-29 cells were transfected with shRNA targeting SH3GLB1 or a negative control, LOVO cells were transfected with OE-RNA targeting SH3GLB1 or a negative control, and SH3GLB1 protein expression was assessed by Western blot. ( E ) CCK-8 assays evaluated cellular growth curves across groups. ( F ) Wound healing assays evaluated the efficacy of migration across groups. ( G ) Colony formation assays evaluated the efficacy of proliferation and stemness across groups. ( H ) Transwell assays evaluated the efficacy of migration across groups. ( I ) Representative images of xenogeneic subcutaneous tumors in HT-29 cells and LOVO cells of BALB/C mice, and comparison of subcutaneous tumor volumes in BALB/C mice inoculated with HT-29 cells and LOVO cells

Article Snippet: Fig. 2 The expression patterns of SH3GLB1 across pan-cancer tissues and its subcellular localization. ( A ) SH3GLB1 expression levels based on the Human Protein Atlas (HPA) database. ( B ) Subcellular localization of SH3GLB1 as derived from the UniProt database. ( C ) Violin plots illustrating variations in SH3GLB1 mRNA expression levels across TCGA-GTEx pan-cancer datasets. ( D ) Paired differential analysis comparing SH3GLB1 mRNA expression levels between tumor and adjacent normal tissues within the TCGA pan-cancer dataset. ( E ) SH3GLB1 gene expression profiles across multiple cancer types: TIMER 3.0 database analysis. ( F ) Boxplots depicting differences in SH3GLB1 protein expression levels using proteomics pan-cancer datasets. ( G ) A heatmap demonstrating SH3GLB1 mRNA expression levels across pan-cancer datasets at the spatial transcriptomic level.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Negative Control, CCK-8 Assay, Migration, Comparison

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3 domain GRB2-like endophilin B1 (SH3GLB1) and superoxide dismutase 2 (SOD2) levels were simultaneously enhanced in the patient derived xenografts (PDX) model of naïve glioblastoma (GBM) tumors after temozolomide (TMZ) (5 mg/kg) treatment for three weeks. ( B ) The results show increased levels of 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA) staining (a reactive oxygen species (ROS) detection probe) in parental U87MG (for 6 h) or A172 (for 24 h) cells after triple co-incubation with TMZ, ATZ, and HNE. H 2 O 2 : 100 μM. Scale bar: 50 μm

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Derivative Assay, Staining, Incubation

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3 domain GRB2-like endophilin B1 (SH3GLB1) is a downstream protein of superoxide dismutase 2 (SOD2). ( A ) The biological network of SH3GLB1-mediated autophagy and SOD2-involved antioxidants was predicted using the STRING bioinformatics tool. ( B ) Resistant cells were transfected with the microtubule-associated protein 1 light chain 3-Enhanced Green Fluorescent Protein (LC3-EGFP) plasmid, and representative fluorescent images for the formation of LC3-EGFP dots (puncta) are shown. Scale bar: 4 μm. ( C ) Protein immunoblotting showing that the resistant cells treated with temozolomide (TMZ) for 24 h induced autophagic reactions, which were attenuated by pretreatment with diethyldithiocarbamic acid (DETC; an SOD inhibitor). ( D ) Immunohistochemical (IHC) staining demonstrating SH3GLB1 expression in primary, recurrent glioblastoma (GBM-R) cells inoculated subcutaneously into mice receiving the indicated treatments for 15 days. A statistical graph is shown in the right-hand panel. Scale bar: 200 μm. ( E ) SOD2 siRNA or ( F ) overexpression vectors were used in U87MG- and A172-resistance cells or U87MG- and A172-parental cells, respectively, three days after transfection to examine the association between SOD2 and SH3GLB1. ( G ) SH3GLB1 siRNA was used in U87MG- and A172-resistance cells, and the association between SH3GLB1 and SOD2 was studied using western blotting. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunohistochemical staining, Immunohistochemistry, Expressing, Over Expression, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: SH3GLB1 levels are regulated by hydrogen peroxide. ( A ) Blots show that co-treatment with TMZ + 4-hydroxynonenal (HNE) + 3-amino-1,2,4-triazole (ATZ), enhanced the expression of SH3GLB1 in parental cells. ( B ) Cells were pretreated with N-acetyl-L-cysteine (NAC) and subjected to three co-treatments. U87MG cells were co-treated for 8 h and A172 cells for 18 h. Intracellular H 2 O 2 levels were measured, and ( C ) SH3GLB1, p-AKT (Ser473), and SOD2 levels were detected by western blotting. ( D ) MK-2206 inhibits p-Akt (Ser473) expression. ( E ) Blots showing the levels of the indicated proteins after co-treatment with TMZ, SC-79 (2-Amino-6-chloro-α-cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester), and NAC. TMZ: 100 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μ, SC-79: 10 μg/mL. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Increased levels of hydrogen peroxide demonstrate different effects on SH3GLB1 expression in the resistant cells. ( A ) MK-2206 was pretreated in the TMZ-treated resistant cells. ( B ) IHC staining demonstrating p-AKT levels in U87MG-R cells transfected with shSH3GLB1 or shControl vectors and inoculated subcutaneously into mice receiving the indicated treatments for 23 days. A statistical graph is shown in the right-hand panel. The arrows indicate the positive staining of SH3GLB1. Scale bar: 200 μm. ( C ) The resistant cells were treated with the indicated reagents. U87MG-R cells were co-treated for 18 h and A172-R cells for 8 h. The levels of intracellular H 2 O 2 were measured. ( D ) Protein immunoblotting after stimulation and rescue treatments. ( E ) Resistant cells were co-treated with TMZ and NAC. TMZ: 100 μM, MK-2206: 5 μM, ATZ: 20 mM, HNE: 10 μM, NAC: 10 mM, MK-2206: 5 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Immunohistochemistry, Transfection, Staining, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) Blots showing the levels of the indicated proteins. Resistant cells were treated with TMZ with or without MK-2206. TMZ: 100 μM, MK-2206: 5 μM ( B ) The parental and resistant cells are treated with increasing concentrations of extracellular H 2 O 2 for 24 h. H 2 O 2 concentrations are indicated. The summary graph demonstrates the difference in the expression of SH3GLB1 in response to extracellular H 2 O 2 between the two cell lines. ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ combined with the inhibitors of an H 2 O 2 -related enzyme affects autophagy levels and tumor growth in the resistant cells. ( A ) The triple co-treatment caused simultaneous changes in autophagy and SH3GLB1 expression. ( B ) Proliferation assay results for parental or resistant cells treated with the indicated compounds are shown as bar graphs, suggesting that the resistant cells were more susceptible to H 2 O 2 accumulation. ( C ) Morphology of A172 and A172-R cells after 72 h of treatment. Control group is no TMZ-treated group. Scale bar: 100 μm. ( D ) Arrows indicate the inhibition of SH3GLB1 levels in the TMZ+HNE and TMZ+HNE+ATZ groups. ( E ) The mice were subcutaneously injected with luciferase-expressing U87MG-R cells. Bioluminescence signals were recorded on the indicated days using an IVIS imaging system. The growth curves of the tumors were analyzed according to the bioluminescence intensity. (N = 5 in each group) ( F ) Immunoblots showing the protein levels of xenograft tumor lysates from mice harvested after the indicated treatments. TMZ: 5 mg/kg, HNE: 2.5 mg/kg ( G ) The IHC staining demonstrates autophagy levels in shSH3GLB1 or shControl group of U87MG-R cells subcutaneously injected in mice and those receiving the indicated treatments. The statistic graph is shown in the right panel. Scale bar: 200 μm. Scale bar in the enlarged graph represents 1 mm. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3~5 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Proliferation Assay, Control, Inhibition, Injection, Luciferase, Imaging, Western Blot, Immunohistochemistry

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: Mitochondrial dysfunction affects SH3GLB1 expression via H 2 O 2 /AKT signaling. ( A ) H 2 O 2 levels in resistant cells were measured after the indicated treatments. TMZ: 100 μM, CCCP: 10 μM ( B ) The protein immunoblot shows that SH3GLB1 levels are regulated by treating with CCCP for 24 h with or without TMZ. Resistant cells (U87MG-R) were treated with TMZ with or without HgCl 2 . ( C ) The statistic graph shows H 2 O 2 levels in the indicated treatments. ( D ) The blots demonstrate the indicated protein expression after the treatments. The statistic graphs are shown. TMZ: 100 μM, HgCl 2 : 20 μM. For each blot, the adjacent bar chart depicts the fold change relative to control. N = 3 in each group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: ( A ) SH3GLB1 and Bax levels were compared between normal and GBM tissues from the TCGA-GBM dataset. ( B ) The protein immunoblots showed levels of Bax-α (21 kd; the lower arrow) and Bax-β (24 kd; the upper arrow) in the parental cells and the derived resistant cells. Corresponding fold-change values (relative to control) are shown in the lower box. ( C ) The western blotting showed that the resistant cells (A172-R) were treated with TMZ with or without HgCl 2 . TMZ: 100 μM, HgCl 2 : 20 μM. Corresponding fold-change values (relative to control) are shown beneath the Western blot panels. N = 3 in each group

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Western Blot, Derivative Assay, Control

Journal: Oncology Research

Article Title: Revealing the Roles of the SH3GLB1-Hydrogen Peroxide Axis in Glioblastoma Multiforme Cells

doi: 10.32604/or.2025.071258

Figure Lengend Snippet: TMZ elevates reactive oxygen species (ROS) levels, including H 2 O 2 . In resistant GBM cells, moderate H 2 O 2 activates AKT, driving SH3GLB1 expression and sustaining drug resistance, whereas excessive H 2 O 2 (e.g., following HNE co-treatment) suppresses SH3GLB1 and impairs resistance. Furthermore, HgCl 2 downregulates mitochondrial aquaporin-9 (AQP9), reducing H 2 O 2 flux and SH3GLB1 expression. This schematic illustrates how H 2 O 2 levels, AKT activation, and AQP9 modulation collectively shape SH3GLB1-driven resistance

Article Snippet: The following antibodies were used for western blot analyses: SH3GLB1 (Cat No. 15422-1-AP; 1:5000), aquaporin 9 (AQP9; Cat No. 20380-1-AP; 1:5000) (Proteintech, Rosemont, IL, USA), Microtubule-associated protein 1 light chain 3B (LC3B; Santa Cruz Biotechnology, Dallas, TX, USA; sc-376404; 1:20000), p62 (Cat No. 5114; 1:6000), SOD2 (Cat No. 13141; 1:6000), AKT (Cat No. 9272; 1:6000), p-AKT (Cat No. 9271; 1:6000) (Cell Signaling, Danvers, MA, USA), vinculin (Thermo Fisher Scientific, Waltham, MA, USA; Cat No. 14-9777-82; 1:10000), and actin (Merck Millipore, Burlington, MA, USA; Cat No. MAB1501; 1:20000).

Techniques: Expressing, Activation Assay