mouse anti fn1  (Santa Cruz Biotechnology)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Metabolic Reprogramming Into a Glycolysis Phenotype Induced by Extracellular Vesicles Derived From Prostate Cancer Cells

doi: 10.1016/j.mcpro.2025.100944

Figure Lengend Snippet: Quantitative proteomics of PC-3 and PC-3AcT EVs. A , comparison of identified EV proteins with Vesiclepedia ( www.microvesicles.org ). Venn diagram indicated that the majority of both EV proteins were previously known EV proteins. B , most of top 100 EV proteins frequently identified in Vesiclepedia are included in our EV proteome. C , heatmap showing enrichment of EV marker proteins related to endosome, tetraspanins, membrane-binding proteins, and EV-associated proteins, which are canonical EV proteins by MISEV guidelines . D , differentially regulated proteins in PC-3 and PC-3AcT EVs are indicated in a volcano plot. Representative proteins validated by Western blotting are indicated by a green arrow. E , Western blotting showing differential expression of proteins including CTNNB1, PKM, FN1, and TSG101 as quantified in proteomics. Additionally, CTNNB1 was further validated in independently isolated EVs to confirm its differential expression observed in quantitative proteomics ( supplemental Fig. S4 ). Western blotting analyses were performed using independent biological replicate of isolated EVs, separate from those used in proteomics. F , GO analyses for biological process, cellular component, and molecular function in significant and nonsignificant regulated proteins between PC-3 and PC-3AcT EVs ( p -value <0.1).

Article Snippet: The following primary antibodies were used: rabbit anti-CD81 (dilution 1:1000, 56039S), rabbit anti-hexokinase 1 (HK1) (1:500, 2024S), rabbit anti-hexokinase 2 (HK2) (1:500, 2887), rabbit anti-phosphofructokinase (PFKP) (1:500, 8164), rabbit anti-p-ERK (1:500, 9101), rabbit anti-ERK (1:500, 9102S), rabbit anti-cyclin B1 (1:500, 12,231), rabbit anti-p-cdc42 (Thr161) (1:500, 9114S), rabbit anti-p-cdc2 (Tyr15) (1:500, 4539), mouse anti-β-actin (1:1000, 3700S) purchased from Cell Signaling Technology; mouse anti-CD9 (1:1000, MAB2529), mouse anti-fibronectin (FN1) (1:500, MAB1918) purchased from R&D Systems; mouse anti-cytochrome c (CYCS) (1:1000, 556,433) purchased from BD; rabbit anti-syntenin-1 (1:1000, ab19903), mouse anti-TSG101 (1:1000, ab125011) purchased from Abcam; mouse anti-β-catenin (CTNNB1) (1:500, sc-7963), rabbit anti-cyclin D1 (1:500, sc-718), mouse anti-pyruvate kinase M1/2 (PKM) (1:500, sc-365684) purchased from Santa Cruz Biotechnology; and OXPHOS Human WB antibody Cocktail (mouse anti-ATP5A, -UQCRC2, -SDHB, -COX2, -NDUFB8) (1:500, 45-8199) purchased from Invitrogen.

Techniques: Quantitative Proteomics, Comparison, Marker, Membrane, Binding Assay, Western Blot, Isolation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Metabolic Reprogramming Into a Glycolysis Phenotype Induced by Extracellular Vesicles Derived From Prostate Cancer Cells

doi: 10.1016/j.mcpro.2025.100944

Figure Lengend Snippet: Increased uptake of PC-3AcT EVs and regulated proteins related to their uptake by PC-3 cells. A , far red –labeled EVs at 2.0 × 10 9 particles/ml were used to treat PC-3 cells and their taken fluorescent EVs were measured by fluorescence microscope. PC-3AcT EVs showed better uptake than PC-3 EVs in PC-3 cells. Please note that fluorescent spots that exceed 1 μm represent clusters of labeled EV-derived components or aggregates in cellular compartments ( e.g. , endosomes or lysosomes). B , differentially regulated EV proteins were mapped in KEGG ECM–receptor interaction annotation. Collagen COLA1, FN1, and other ECMs were overexpressed in PC-3AcT EVs. C , protein–protein interaction network among 1.5-fold upregulated PC-3AcT EV proteins was mapped in the BioGRID protein interaction database and their interactions were visualized by Cytoscape ( cytoscape.org ). EV proteins were grouped based on their main functionality and related proteins as indicated by a dashed line box. p -values (∗∗∗∗ < 0.0001).

Article Snippet: The following primary antibodies were used: rabbit anti-CD81 (dilution 1:1000, 56039S), rabbit anti-hexokinase 1 (HK1) (1:500, 2024S), rabbit anti-hexokinase 2 (HK2) (1:500, 2887), rabbit anti-phosphofructokinase (PFKP) (1:500, 8164), rabbit anti-p-ERK (1:500, 9101), rabbit anti-ERK (1:500, 9102S), rabbit anti-cyclin B1 (1:500, 12,231), rabbit anti-p-cdc42 (Thr161) (1:500, 9114S), rabbit anti-p-cdc2 (Tyr15) (1:500, 4539), mouse anti-β-actin (1:1000, 3700S) purchased from Cell Signaling Technology; mouse anti-CD9 (1:1000, MAB2529), mouse anti-fibronectin (FN1) (1:500, MAB1918) purchased from R&D Systems; mouse anti-cytochrome c (CYCS) (1:1000, 556,433) purchased from BD; rabbit anti-syntenin-1 (1:1000, ab19903), mouse anti-TSG101 (1:1000, ab125011) purchased from Abcam; mouse anti-β-catenin (CTNNB1) (1:500, sc-7963), rabbit anti-cyclin D1 (1:500, sc-718), mouse anti-pyruvate kinase M1/2 (PKM) (1:500, sc-365684) purchased from Santa Cruz Biotechnology; and OXPHOS Human WB antibody Cocktail (mouse anti-ATP5A, -UQCRC2, -SDHB, -COX2, -NDUFB8) (1:500, 45-8199) purchased from Invitrogen.

Techniques: Labeling, Fluorescence, Microscopy, Derivative Assay

Journal: Cell Death & Disease

Article Title: Knockout of integrin αvβ6 protects against renal inflammation in chronic kidney disease by reduction of pro-inflammatory macrophages

doi: 10.1038/s41419-024-06785-5

Figure Lengend Snippet: A Representative immunostaining images and quantification of integrin αvβ6 in kidney sections from sham and uIRI mice on days 1, 3, 7, and 14 (scale bar, 100 μm). The boxed area in the uIRI-14d panel is magnified in the right panel (scale bar, 50 μm). ( n = 4-5 per group). B Correlations between integrin αvβ6 staining quantification and renal inflammation of sham, uIRI-7d, and uIRI-14d mice. For renal inflammation, see Fig. S , it is indicated by Tnf , Il1b , Ccl2 , and Il6 relative mRNA levels detected by qPCR ( n = 14). C Relative mRNA levels of renal inflammatory factors ( Tnf , Il1b , Ccl2 , and Il6 ) in WT and Itgb6 -/- mice were detected by qPCR ( n = 3–5 per group). D , E Neutrophils, dendritic cells, T cells, B cells, and macrophages in kidneys of sham or uIRI-7d mice were detected by flow cytometry and compared. The gating strategies and representative images of different immune cells are shown in Fig. S ( n = 3–5 per group). F Comparisons of anti-inflammatory (Ly6c - F4/80 hi ) or pro-inflammatory (Ly6c + F4/80 int ) macrophages in kidneys of sham or uIRI-7d mice ( n = 3–5 per group). G Western blot of α-SMA and fibronectin in sham or uIRI-7d kidneys. The quantifications of the relative levels of α-SMA/α-Tubulin and fibronectin/α-Tubulin are shown ( n = 3 per group). H Representative images and quantifications of Sirius Red staining in kidney sections from sham or uIRI-7d mice (scale bar, 100 μm) ( n = 3–6 per group). I Serum BUN and creatinine in control or AAN mice. ( n = 3 per group). J Relative mRNA levels of renal inflammatory factors ( Tnf , Il1b , Ccl2 , and Il6 ) in control or AAN mice were detected by qPCR ( n = 3 per group). K Comparison of pro-inflammatory (Ly6c + F4/80 int ) macrophages in control or AAN mice kidneys ( n = 3 per group). L Western blot of α-SMA and fibronectin in control or AAN mice kidneys. The quantifications of the relative levels of α-SMA/α-Tubulin and fibronectin/α-Tubulin are shown ( n = 3 per group). M Representative images and quantifications of Sirius Red staining in kidney sections from control or AAN mice (scale bar, 100 μm) ( n = 3 per group). Data are presented as mean ± SEM of three biological replicates. One-way ANOVA ( A , C – M ), and Pearson correlation test ( B ) were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The antibodies used in western blot were as follows: goat anti-mouse integrin β6 antibody (RD, AF2389), mouse anti-mouse α-SMA Ab (Sigma-Aldrich, A5228), rabbit anti-mouse Fibronectin Ab (BOSTER, BA1772), sheep anti-mouse IL-34 Ab (RD, AF5195), rabbit anti-mouse YAP Ab (CST, 14074 S), rabbit anti-mouse p-YAP (S127) Ab (CST, 4911 S), mouse anti-mouse GAPDH Ab (Abcam, ab8245), mouse anti-mouse α-Tubulin Ab (CST, 12351 S), and mouse anti-mouse β-actin Ab (Abcam, ab8226).

Techniques: Immunostaining, Staining, Flow Cytometry, Western Blot, Control, Comparison

Journal: Cell Death & Disease

Article Title: Knockout of integrin αvβ6 protects against renal inflammation in chronic kidney disease by reduction of pro-inflammatory macrophages

doi: 10.1038/s41419-024-06785-5

Figure Lengend Snippet: A Immunofluorescence staining for different segment-specific tubular markers (green), integrin αvβ6 (red), and DAPI (blue) in WT-uIRI-7d kidney sections (scale bar, 50 μm). Segment-specific tubular markers were used as follows: proximal tubule, LTL; distal tubule, PNA; and collecting duct, DBA. B Immunofluorescence staining for KIM-1 (green), integrin αvβ6 (red), and DAPI (blue) in uIRI-7d kidney sections (scale bar, 50 μm). C Schematic of generating Itgb6 fl/fl Pepck-Cre or Itgb6 fl/fl mice which delete αvβ6 specific in PTCs or not and experimental design. D Relative mRNA levels of renal inflammatory factors ( Tnf , Il1b , Ccl2 , and Il6 ) in Itgb6 fl/fl Pepck-Cre - or Itgb6 fl/fl -uIRI-7d mice were detected by qPCR ( n = 4-5 per group). E Comparison of pro-inflammatory (Ly6c + F4/80 int ) macrophages in Itgb6 fl/fl Pepck-Cre - or Itgb6 fl/fl -uIRI-7d mice kidneys ( n = 4-5 per group). F Western blot of α-SMA and fibronectin in Itgb6 fl/fl Pepck-Cre - or Itgb6 fl/fl -uIRI-7d mice kidneys. The quantifications of the relative levels of α-SMA/α-Tubulin and fibronectin/α-Tubulin are shown ( n = 4-5 per group). G Representative images and quantifications of Sirius Red staining in kidney sections from Itgb6 fl/fl Pepck-Cre - or Itgb6 fl/fl -uIRI-7d mice (scale bar, 100 μm) ( n = 4-5 per group). H Representative immunostaining images and correlation analysis of integrin αvβ6 and F4/80 in kidney serial sections from WT-uIRI-7d mice (scale bar, 50 μm). Black triangles indicate αvβ6-expressing tubules in serial sections ( n = 56 fields from 6 mice). I Schematic of the in vitro co-culture system. TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then co-cultured with RAW264.7 cells for 12 h. J , K Migration and pro-inflammatory differentiation of RAW264.7 cells after co-culturing with si-NC or si- Itgb6 -transfected TKPTS cells under the hypoxic stimulation or not (scale bar, 25 μm). Pro-inflammatory differentiation is indicated by relative mRNA levels of inflammatory factors ( Tnf , Il1b Ccl2 , and Il6 ) ( n = 3 per group). Data are presented as mean ± SEM of three biological replicates. Student’s t -test ( D – G ), Pearson correlation test ( H ), and one-way ANOVA ( J , K ) were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: The antibodies used in western blot were as follows: goat anti-mouse integrin β6 antibody (RD, AF2389), mouse anti-mouse α-SMA Ab (Sigma-Aldrich, A5228), rabbit anti-mouse Fibronectin Ab (BOSTER, BA1772), sheep anti-mouse IL-34 Ab (RD, AF5195), rabbit anti-mouse YAP Ab (CST, 14074 S), rabbit anti-mouse p-YAP (S127) Ab (CST, 4911 S), mouse anti-mouse GAPDH Ab (Abcam, ab8245), mouse anti-mouse α-Tubulin Ab (CST, 12351 S), and mouse anti-mouse β-actin Ab (Abcam, ab8226).

Techniques: Immunofluorescence, Staining, Comparison, Western Blot, Immunostaining, Expressing, In Vitro, Co-Culture Assay, Transfection, Cell Culture, Migration

Journal: Cell Death & Disease

Article Title: Knockout of integrin αvβ6 protects against renal inflammation in chronic kidney disease by reduction of pro-inflammatory macrophages

doi: 10.1038/s41419-024-06785-5

Figure Lengend Snippet: A TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then the relative mRNA levels of macrophage activators in TKPTS cells were detected with qPCR and shown with heatmap ( n = 3 per group). B Western blot of IL-34 in si-NC- or si- Itgb6 -transfected hypoxic TKPTS cells. The quantification of the relative levels of IL-34/β-actin is shown ( n = 4 per group). C TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then IL-34 in supernatant was detected by ELISA ( n = 3 per group). D , E Western blot and immunostaining of IL-34 in uIRI-7d or AAN kidneys from WT and Itgb6 -/- mice (scale bar, 50 μm). The quantification of the relative levels of IL-34/α-Tubulin and quantification of IL-34 positive area proportion are shown ( n = 3–5 per group). F Immunofluorescence staining for IL-34 (red), KIM-1 (green), and DAPI (blue) in WT-uIRI-7d kidney sections (scale bar, 50 μm). G Schematic of an in vitro rescue experiment. TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then co-cultured with RAW264.7 cells for 12 h. RmIL-34 or vehicle was supplemented into the co-culture system as indicated. H , I Migration and pro-inflammatory differentiation of RAW264.7 cells in the co-culture system (scale bar, 25 μm) ( n = 3-4 per group). J Schematic of another in vitro reverse rescue experiment. TKPTS cells transfected with oe-NC or oe- Itgb6 plasmids were pre-stimulated with hypoxia for 24 h or not, and then co-cultured with RAW264.7 cells for 12 h. Anti-IL-34 antibody (αIL-34) or isotype control (10 μg/mL) was added into the co-culture system as indicated. K , L Migration and pro-inflammatory differentiation of RAW264.7 cells in the co-culture system (scale bar, 25 μm) ( n = 3 per group). M Schematic of an in vivo rescue experiment. Itgb6 -/- mice were intraperitoneally supplemented with recombinant mouse IL-34 (rmIL-34) or vehicle (1 μg/dose) immediately and 3 days after uIRI, and WT-IRI-7d mice received vehicle served as controls. N Relative mRNA levels of renal inflammatory factors ( Tnα , Il1b , Ccl2 , and Il6 ) in WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle were detected by qPCR ( n = 4-5 per group). O Comparison of pro-inflammatory (Ly6c + F4/80 int ) macrophages in WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle ( n = 4-5 per group). P Western blot of α-SMA and fibronectin in kidneys of WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle. The quantifications of the relative levels of α-SMA/α-Tubulin and fibronectin/α-Tubulin are shown ( n = 3 per group). Q Representative images and quantifications of Sirius Red staining in kidney sections from WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle (scale bar, 100 μm) ( n = 4-5 per group). Data are presented as mean ± SEM of three biological replicates. Student’s t -test ( B , D , E , K , L ) and one-way ANOVA ( C , H – Q ) were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. # p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001; compared with si- Itgb6 + rmIL-34 group (0 ng/mL) ( H , I ); # p < 0.05; ## p < 0.01; up-regulated degree by oe- Itgb6 was compared between isotype (light blue background) and αIL-34 group (light orange background) ( K , L ); ns, not significant.

Article Snippet: The antibodies used in western blot were as follows: goat anti-mouse integrin β6 antibody (RD, AF2389), mouse anti-mouse α-SMA Ab (Sigma-Aldrich, A5228), rabbit anti-mouse Fibronectin Ab (BOSTER, BA1772), sheep anti-mouse IL-34 Ab (RD, AF5195), rabbit anti-mouse YAP Ab (CST, 14074 S), rabbit anti-mouse p-YAP (S127) Ab (CST, 4911 S), mouse anti-mouse GAPDH Ab (Abcam, ab8245), mouse anti-mouse α-Tubulin Ab (CST, 12351 S), and mouse anti-mouse β-actin Ab (Abcam, ab8226).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining, Immunofluorescence, Staining, In Vitro, Cell Culture, Co-Culture Assay, Migration, Control, In Vivo, Recombinant, Comparison