p enos  (Cell Signaling Technology Inc)

Journal: Clinical and experimental hypertension (New York, N.Y. : 1993)

Article Title: Anthocyanin attenuates pulmonary arterial hypertension and associated heart failure via improving mitochondrial function through Nrf2-dependent mechanism.

doi: 10.1080/10641963.2025.2503805

Figure Lengend Snippet: Figure 2. Anthocyanin improved endothelial function of pulmonary artery in mice exposed to hypoxia. C57BL/6 mice were subcutaneously injected with 20 mg/kg Sugen5416 once a week and were exposed to hypoxia (10% O2) or normoxia with anthocyanin (10 mg/kg/day) or vehicle (sterile water) treatment by oral gavage for 21 days. (a-c) phe-induced contraction, ach-induced relaxation and SNP-induced relaxation in pulmonary artery from the mice exposed to hypoxia or normoxia. (d) The phosphorylation and expression of eNOS at Ser1177 in pulmonary artery from the mice exposed to hypoxia or normoxia. Data are expressed as the means ± SD (n = 5/ group). *p < .05 vs others. N+V, Normoxia+Vehicle; N+A, Normoxia+Anthocyanin; H+V, Hypoxia+Vehicle; H+A, Hypoxia+Anthocyanin.

Article Snippet: The PVDF membranes were incubated with primary antibodies, including phosphorylated eNOS at Ser1177 (1:500, Cell Signaling Technology, Danvers, MA, USA), t-eNOS (1:1000, Cell Signaling Technology), Nrf2 (1:300; Abcam, Cambridge, UK), H3 (1:300; Abcam), GAPDH (1:500, Abcam), over night at 4°C, and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Injection, Sterility, Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: Sialin2-mediated endosomal PI3K-AKT-eNOS signaling promotes nitrate-induced NO production and vascular function a–d, IF staining images and quantification of pAKT S473 (a, b) or peNOS S1177 (c, d) in sg SLC17A5 HUVEC cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. e, f, Images (e) and quantification (f) of the 12 and 24 h tube formation ability in sg SLC17A5 HUVEC cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 3. g, h, Crystal violet (CV) viability assay (g), and cell death detection (h) in sg SLC17A5 HUVEC cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Date normalized to control. N = 3 from three independent experiments, each in triplicate. i, Quantification of nitric oxide (NO) production in sg SLC17A5 HUVEC cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 3. j, k, PLA of Sialin/Sialin2-pAKT T308 (j) or Sialin/Sialin2-pAKT S473 (k) in nitrate- treated (4 mM, 4 h) HUVEC cells with or without chlorpromazine (CPZ, 6 μg/ml, 30 min). N = 30 cells from representative experiments of three repeats. l, IF staining images and quantitation of peNOS S1177 with EEA1 in OE-Sialin2 HUVEC cells treated with CPZ (6 μg/ml, 30 min) and miransertib (10 μM, 24 h). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. m, NO production in OE-Sialin2 HUVEC cells treated with chlorpromazine (CPZ, 6 μg/ml, 30 min) and miransertib (10 μM, 24 h). N = 3. n, Quantification of the 12 h tube formation ability in control and OE-Sialin2 HUVEC cells treated with nitrate (4 mM, 4 h) after pretreated with L-NAME (500 μM, 3 h). N = 3. See images in . o–r, Systolic blood pressure (SBP; o), mean blood pressure (MBP; p), diastolic blood pressure (DBP; q), and NO production (r) in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) treated with nitrate (2.25 mmol/kg) after pretreated with miransertib (10 mg/kg, 2 h) or L-NAME (20 mg/kg, 30 min), or with nitroglycerin (GTN, 0.5 mg/kg) alone. N = 8. s–u, IF staining images and quantification of peNOS S1177 in thoracic aorta (s, t) and mesenteric artery (u) from WKY rats and SHRs treated with nitrate (2.25 mmol/kg) after pretreated with miransertib (10 mg/kg, 2 h) or L-NAME (20 mg/kg, 30 min), or with nitroglycerin (GTN, 0.5 mg/kg) alone. N = 16 from eight rats. See mesenteric artery images in . v, Schematic illustration of nitrate activating endosomal PI3K-AKT-eNOS signaling via Sialin2-LYN-EGFR to promote NO production in endothelial cells, alleviate endothelial cell dysfunction, and reduce hypertension. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm or 200 μm (e and t).

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Staining, Viability Assay, Control, Quantitation Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Immunoblot analysis of peNOS S1177 , eNOS, pAKT T308 , pAKT S473 , and AKT in sg SLC17A5 HUVEC cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. b–e, Methyl thiazolyl tetrazolium (MTT) viability assay (b, c), crystal violet (CV) viability assay (d), and cell death detection (e) in sg SLC17A5 HUVEC and HeLa cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Date normalized to control. N = 3 from three independent experiments, each in triplicate. f, g, Caspase-3 activity assay in sg SLC17A5 HUVEC (f) and HeLa (g) cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Activity normalized to control. N = 3. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Western Blot, MTT Viability Assay, Viability Assay, Control, Caspase-3 Activity Assay, Activity Assay

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a–d, PLA of Sialin/Sialin2-pAKT T308 (a, b) or Sialin/Sialin2-pAKT S473 (c, d) in nitrate-treated (4 mM, 4 h) HeLa and NRK cells with or without CPZ (6 μg/ml, 30 min). N = 30 cells from representative experiments of three repeats. e, IF staining images and quantitation of pAKT S473 with EEA1 in OE-Sialin2 HUVEC cells treated with CPZ (6 μg/ml, 30 min). Colocalization quantified by Pearson’s correlation coefficient. N = 30 cells from representative experiments of three repeats. f, Immunoblot analysis of peNOS S1177 , eNOS, pAKT T308 , pAKT S473 , and AKT in OE- Sialin2 HUVEC cells treated with CPZ (6 μg/ml, 30 min) or miransertib (10 μM, 24 h). Representative images of n = 3 independent experiments were shown. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Staining, Quantitation Assay, Western Blot

Journal: bioRxiv

Article Title: Sialin2 Senses Nitrate to Activate Endosomal PI3K-AKT- NOS Signaling

doi: 10.1101/2025.05.04.652107

Figure Lengend Snippet: a, Images and quantification of the 12 and 24 h tube formation ability in control and OE-Sialin2 HUVEC cells treated with nitrate (4 mM, 4 h) after pretreated with L- NAME (500 μM, 3 h). N = 3. See quantitation of 12 h in . b–g, MTT viability assay (b, c), CV viability assay (d, e), and cell death detection (f, g) in control and OE-Sialin2 HUVEC or HeLa cells treated with nitrate (4 mM, 4 h) after pretreated with L-NAME (500 μM, 3 h). Date normalized to control. N = 3 from three independent experiments, each in triplicate. h, i, Caspase-3 activity assay in s control and OE-Sialin2 HUVEC (h) or HeLa (i) cells treated with nitrate (4 mM, 4 h) after pretreated with L-NAME (500 μM, 3 h). Activity normalized to control. N = 3. j, IF staining images of peNOS S1177 in mesenteric artery from WKY rats and SHRs treated with nitrate (2.25 mmol/kg) after pretreated with miransertib (10 mg/kg, 2 h) or L-NAME (20 mg/kg, 30 min), or with nitroglycerin (GTN, 0.5 mg/kg) alone. N = 16 from eight rats. See quantitation in . For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 200 μm.

Article Snippet: Monoclonal antibodies against pAKT T308 (Cell Signaling, clone C31E5E, #2965), pAKT S473 (Cell Signaling, clone D9E, #4060), AKT (Cell Signaling, clone C67E7, #4691), p110α (Cell Signaling, clone C73F8, #4249), pEGFR T1068 (Cell Signaling, clone D7A5, #3777), RAB5 (Santa Cruz Biotechnology, clone D-11, #sc-46692; Cell Signaling, clone C8B1, #3547), RAB7 (Santa Cruz Biotechnology, clone B-3, #sc- 376362; Cell Signaling, clone D95F2, #9367), TFR1 (Invitrogen, clone H68.4, #13- 6800), EEA1 (Santa Cruz Biotechnology, clone D-5, #sc-518025), eNOS (Cell Signaling, clone D9A5L, #32027), peNOS S1177 (Cell Signaling, clone C9C3, #9571), HA-tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), Lyn (Proteintech, clone 3C7F2, #60211), ACTB (Proteintech, clone 2D4H5, #66009) , and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), EEA1 (Cell Signaling, #2411), Na,K-ATPase (Cell Signaling, #3010), Lyn (Proteintech, #18135), peNOS S1177 (Invitrogen, #PA5- 104858), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Control, Quantitation Assay, MTT Viability Assay, Viability Assay, Caspase-3 Activity Assay, Activity Assay, Staining