immunoblotting with anti src 3  (Cell Signaling Technology Inc)

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 +/+ ApoE -/- mice exhibit more severe atherosclerosis. (A) SRC-3 was highly expressed in human atherosclerotic plaques. N represents plaque-adjacent vasculature in the lower limb aorta. AS represents atherosclerotic plaques in the lower limb aorta. (B) SRC-3 expression was upregulated in the aortas of ApoE -/- mice after the mice were fed a WD for 12 weeks. (C) SRC-3 was expressed in the endothelial cells and vascular smooth muscle cells of chow-fed SRC-3 -/- ApoE -/- mice and was further increased after the mice were fed a WD for 12 weeks. Sections of frozen aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to X-gal staining. Arrows indicate positively stained cells (blue). Scale bar, 100 µm. (D) SRC-3 promoted atherosclerotic plaque formation. Representative images of en face Oil Red O-stained aortas from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice (left panel). Quantification of the plaque areas in aortas (right panel). (E-K) Cross-sections of the aortic roots from SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice were subjected to (E-F) H&E staining (scale bar, 500 µm (E); scale bar, 200 µm (F)), (G) α-SMA staining (scale bar, 200 µm), (H-I) Masson staining (scale bar, 200 µm (H); scale bar, 200 µm (I)), (J) Oil Red O staining (scale bar, 500 µm), and (K) F4/80 staining (scale bar, 200 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. “×” indicates necrotic area. (L) Plaque stability was significantly increased in SRC-3 -/- ApoE -/- mice. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; ** P <0.01.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 in endothelial cells contributes to the development of atherosclerosis. (A) Western blot showing that AAV-mediated SRC-3 shRNA decreased SRC-3 expression levels in the aortas of ApoE -/- mice. (B) SRC-3 knockdown reduced WD-induced atherosclerotic plaque formation in ApoE -/- mice. Representative images of en face Oil Red O-stained aortas from ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (C-F) Cross-sections of the aortic roots of ApoE -/- mice injected with AAV-mediated SRC-3 shRNA and scramble shRNA were subjected to (C) H&E staining (scale bar, 100 µm), (D) Masson staining (scale bar, 500 µm), (E) Oil Red O staining (scale bar, 100 µm), (F) F4/80 staining (scale bar, 100 µm). Left panels, representative images. Right panels, quantification of stained area or a percentage of lesion area. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Western Blot, shRNA, Expressing, Staining, Injection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 increases ICAM-1 expression during atherosclerosis development. (A) KEGG enrichment pathway analysis and (B) Gene Ontology (GO) biological process analysis of mRNA profiles in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. (C) Selected genes involved in leukocyte recruitment and proinflammatory markers are shown as a heat map. (D) The mRNA level of ICAM-1 in the aortas of SRC-3 -/- ApoE -/- mice was significantly decreased after WD feeding for 12 weeks. (E) The protein level of ICAM-1 in the aortas of SRC-3 +/+ ApoE -/- and SRC-3 -/- ApoE -/- mice after WD feeding for 12 weeks. Each lane represents a pooled sample of three representative mice. (F) Western blot analysis of SRC-3 and ICAM-1 in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta of accident patients. N represents plaque-adjacent vasculature in the lower limb aorta; AS represents atherosclerotic plaques in the lower limb aorta. (G) Correlation between SRC-3 and ICAM-1 protein levels in 16 atherosclerotic plaques and plaque-adjacent vasculature in the lower limb aorta. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: SRC-3 regulates ICAM-1 expression via enhancing NF-κB signaling. (A) The protein levels of SRC-3 and ICAM-1 in SRC-3 siRNA-transfected HUVECs were significantly reduced compared with those in scrambled siRNA-transfected HUVECs after TNFα or IL-1β treatment. (B) The mRNA level of ICAM-1 in the SRC-3 siRNA-transfected HUVECs was markedly decreased after TNFα or IL-1β treatment. (C) ICAM-1 promoter activity was reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment. (D) SRC-3 cooperated with p65 to enhance the activity of the NF-κB reporter (upper panel) and ICAM-1 promoter (lower panel). (E) NF-κB binding site mutation abolished NF-κB-mediated ICAM-1 promoter activity. (F) The recruitment of SRC-3 and p65 was significantly reduced in SRC-3 siRNA-transfected HUVECs after TNFα or IL-1β treatment (right panel). Position of the subfragments detected by ChIP assays (left panel). (G) The SRC-3 siRNA-transfected HUVECs monolayer exhibited a significantly decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (H) Western blot showing ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs. (I) ICAM-1 overexpression in SRC-3 siRNA-transfected HUVECs rescued monocyte attachment to HUVECs and monocyte transendothelial migration after TNFα or IL-1β treatment. The data represent the mean ± SEM of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Expressing, Transfection, Activity Assay, Binding Assay, Mutagenesis, Western Blot, Over Expression, Migration, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Pharmacological inhibition of SRC-3 reduces atherosclerosis. (A) The protein levels of SRC-3, ICAM-1 and p-p65 in bufalin-treated HUVECs were significantly reduced compared with those in vehicle-treated HUVECs after TNFα or IL-1β treatment. The bufalin-treated HUVECs monolayer resulted in a dramatically decreased number of adhered (upper panel) and transmigrated (lower panel) THP-1 cells after TNFα or IL-1β treatment. (B-C) ApoE -/- mice were administered vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (B) Dosing regimen (ApoE -/- prevention model). (C) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (D) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- prevention model. (E-F) ApoE -/- mice were fed a WD for 10 weeks and then treated with vehicle or bufalin (1.0 mg/kg, six times a week) by intraperitoneal injection for 13 weeks concomitant with WD feeding. (E) Dosing regimen (ApoE -/- regression model). (F) Representative images of en face Oil Red O-stained aortas from ApoE -/- mice treated with vehicle or bufalin (left panel). Quantification of the plaque areas of aortas (right panel). The data represent the mean ± SEM. (G) The protein levels of SRC-3 and ICAM-1 in the aortas of ApoE -/- mice treated with bufalin were significantly reduced in the ApoE -/- regression model. The data represent the mean ± SEM. The results are representative of three independent experiments. P values were calculated by unpaired two-tailed Student's t-test. *, P <0.05; **, P <0.01;***, P <0.001.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Inhibition, Injection, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: SRC-3 deficiency prevents atherosclerosis development by decreasing endothelial ICAM-1 expression to attenuate macrophage recruitment

doi: 10.7150/ijbs.74864

Figure Lengend Snippet: Schematic model of the mechanism by which SRC-3 accelerates atherosclerosis development. SRC-3 promotes atherosclerosis development by increasing ICAM-1 transcription by enhancing the function of NF-κB in endothelial cells to promote macrophage recruitment. SRC-3 depletion or pharmacological inhibition of SRC-3 by bufalin ameliorates atherosclerosis development through decreasing endothelial ICAM-1 expression and macrophage recruitment via reduction of NF-κB function.

Article Snippet: Equal amounts of proteins were loaded onto 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore), followed by immunoblotting with anti-SRC-3 (Cell Signaling Technology, 5E11, #2116), anti-human ICAM-1 (Abcam, EPR4776, ab109361), anti-mouse ICAM-1 (Abcam, EPR16608, ab179707), anti-p65 (Cell Signaling Technology, D14E12, #8284), anti-p-p65 (Cell Signaling Technology, Ser536, #3031), anti-GAPDH (Cell Signaling Technology, D16H11, #5174) and anti-β-actin (Sigma, AC-15, #A5441).

Techniques: Inhibition, Expressing