primary antibody spns2  (GeneTex)

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: Spinster homolog 2 (SPNS2) expression is reduced in aging vascular and senescent endothelial cells (ECs) and has a negative correlation with aging degree. a Expression of SPNS2 in human carotid artery from GSE100927 (healthy carotid artery, n = 12, atherosclerosis carotid artery, n = 29, two-sided t -test). Source data are provided as a Source Data file. b , c mRNA levels of KI67 and Lamin B1 b and SPNS2 c in the aorta of mice during different months. Data are presented as the mean ± standard error of mean (SEM) ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. d Immunofluorescence of Lamin B1, SPNS2 and CD31 in the aorta of mice during different months. The cell nucleus exhibits blue fluorescence (DAPI), Scale bar, 100 μm. e Regression analysis of KI67 and LaminB1 expression data with SPNS2 expression from the aorta of mice during different months. f SA-β-gal staining of control human umbilical vein endothelial cell (HUVEC) and HUVECs treated with H 2 O 2 . Scale bar, 200 μm. g Immunoblot analysis of P53 and Lamin B1 in control HUVECs and HUVECs treated with H 2 O 2 . h Immunoblot analysis of SPNS2 in HUVECs and HUVECs treated with H 2 O 2 . i mRNA levels of SPNS2 in HUVECs and HUVECs treated with H 2 O 2 . Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test.

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency in mice ECs aggravates vascular dysfunction and collagen deposition. a, b Immunofluorescence of SPNS2, Lamin B1 and CD31 in the aorta of SPNS2 flox/flox and SPNS2 TEK−/− mice. The cell nucleus exhibits blue fluorescence (DAPI), Scale bar, 100 μm. c mRNA levels of SPNS2, KI67, Lamin B1, P21, MMP3, IL-6, IL-1β, and IL-8 in SPNS2 flox/flox and SPNS2 TEK−/− mice. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. d , e Small animal diagnostic ultrasound of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice. Blood flow analysis in SPNS2 flox/flox and SPNS2 TEK−/− mice. f Masson staining of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice quantified using ImageJ. Scale bar, 100 μm. No adjustments. g Sirius red staining of aorta from SPNS2 flox/flox and SPNS2 TEK−/− mice quantified using ImageJ. Scale bar, 100 μm. No adjustments

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Immunofluorescence, Fluorescence, Diagnostic Assay, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency aggravates HUVEC senescence. a Immunoblot analysis of SPNS2 in control, Sh-negative control (NC), and Sh-SPNS2 HUVECs. b Morphology of control, Sh-NC, and Sh-SPNS2 HUVECs by crystal violet staining and quantified by Image J. Scale bar, 200 μm. c The union of differential genes of all comparative combinations of Sh-NC and Sh-SPNS2 HUVECs. d Volcano plot showing fold changes in translation levels between Sh-NC and Sh-SPNS2 HUVECs. The upregulated (red) and downregulated (green) genes are highlighted. Source data are provided as a Source Data file. e KEGG analysis of the RNAseq data from HUVECs with SPNS2 knockdown. f SA-β-gal staining of control, Sh-NC, and Sh-SPNS2 HUVECs. Scale bar, 200 μm. g MTT assay detects cell viability of control, Sh-NC, and Sh-SPNS2 HUVECs. h Immunofluorescence of control, Sh-NC, and Sh-SPNS2 HUVECs. The cell nucleus exhibits blue fluorescence (DAPI), and KI67 exhibits yellow fluorescence (RFP). Scale bar, 200 μm. Enlarged image on the left. Scale bar, 50 μm. i Immunoblot analysis of Lamin B1 and P53 in control, Sh-NC, and Sh-SPNS2 HUVECs. j mRNA levels of Il-1β, Il-6, Il-8, and Mmp3 in control, Sh-NC, and Sh-SPNS2 HUVECs. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t -test. k Immunoblot of SPNS2 in control, OE-negative control (NC), and OE-SPNS2 HUVECs. l mRNA levels of Il-1β, Il-6, Il-8, and Mmp3 in control, OE-NC, and OE-SPNS2 HUVECs. Data are presented as the mean ± SEM ( n = 10). ns, not significant, * P < 0.05, ** P < 0.01, two-sided t-test.

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Western Blot, Control, Negative Control, Staining, Knockdown, MTT Assay, Immunofluorescence, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency induces HUVEC senescence via mitochondrial function damage. a Transmission electron microscopic image of control, Sh-NC, and Sh-SPNS2 HUVECs. Mitochondria are highlighted by arrows, 500 nm. b Transmission electron microscopic image of control, Sh-NC, and Sh-SPNS2 HUVECs. Mitochondria are stained by Mitored (red), 10 μm. c Immunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in control, Sh- NC, and Sh-SPNS2 HUVECs d N, N, N′, N′-tetramethyl-ethylenediamine (TMRE) staining of control, Sh-NC, and Sh-SPNS2 HUVECs measured using flow cytometry. e DCFH-DA staining of control, Sh-NC, and Sh-SPNS2 HUVECs measured using flow cytometry. f TMRE staining of Sh-SPNS2 HUVECs and Sh-SPNS2 HUVECs treated with NAC measured using flow cytometry. g DCFH-DA staining of Sh-SPNS2 HUVECs and Sh-SPNS2 HUVECs treated with NAC measured using flow cytometry. h SA-β-gal staining of control, Sh-NC, Sh-SPNS2, and Sh-SPNS2 HUVECs treated with NAC. Scale bar, 200 μm. i I mmunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in control HUVECs and HUVECs treated with NAC. j Immunoblot analysis of Lamin B1 in HUVECs and HUVECs treated with NAC. k IL-6, IL-1β and IL-8 protein level of HUVECs and HUVECs treated with NAC were detected using ELISA kit

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Transmission Assay, Control, Staining, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: SPNS2 deficiency impairs mitochondrial function by promoting PKM-mediated pyruvate metabolism dysregulation. a Heatmap analysis shows the relative expression levels of pyruvate metabolism genes from GO enrichment. b Heatmap analysis shows the relative expression levels of HIF-1 signaling pathway genes from KEGG enrichment. c Immunoblot analysis of HIF1α in control, Sh-negative control (NC), and Sh-SPNS2 HUVECs. d , e , f , g , h Contents of pyruvate, ethanol, lactic acid, ATP, and acetyl Co-A in control, Sh-NC, and Sh-SPNS2 HUVECs. i Immunoblot analysis of PKM in control, Sh-NC, Sh-SPNS2 HUVECs, Sh-SPNS2 HUVECs treated with nc-siRNA, and Sh-SPNS2 HUVECs treated with PKM-siRNA. j , k , l Contents of pyruvate, ethanol, and lactic acid in Sh-SPNS2 HUVECs treated with nc-siRNA and those treated with PKM-siRNA. m Immunoblot analysis of PGC-1α, HSP60, VDAC1 and TOM20 in Sh-SPNS2 HUVECs treated with nc-siRNA and HUVECs treated with PKM-siRNA. n , o Contents of ATP and acetyl Co-A in Sh-SPNS2 HUVECs treated with nc-siRNA and Sh-SPNS2 HUVECs treated with PKM-siRNA

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: Expressing, Western Blot, Control, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

doi: 10.1186/s12964-024-01859-5

Figure Lengend Snippet: Primers for qPCR

Article Snippet: After sealing with 5% skim milk from TBST for 1.5 h, the membrane was incubated at 4 °C 10–12 h with the corresponding primary antibody SPNS2 (1:2000, GeneTex, USA), P53 (1;5000, Proteintech, UK), Lamin B1 (1:5000, Proteintech), PKM (1:1000, Proteintech), and β-actin (1:5000, Proteintech).

Techniques: