Western blot and immunoprecipitation analyses were performed as previously described56 (link). For western blot analysis, the following primary antibodies were used: Nogo-B (#AF-6034; R&D, Minneapolis, MN, dilution 1:1,000); eNOS, SPTLC1 and Hsp90 (#610297, #611304 and #610419; BD, Biosciences, San Jose, CA; dilution 1:1,000); nNOS and phospho-S1176-eNOS (#C12H1 and #9571s respectively; Cell Signaling Technology, Danvers, MA; dilution 1:1,000); anti-HA (#11867423001; Roche, Nutley, NJ; dilution 1;1,000); S1P1, c-Myc, ORMDL1/2/3, VE-cadherin and NgBR (#sc-25489, 1:2,000; #sc-40, 1:1,000; #sc-161143, #sc-6458, 1:1,000; and #sc138044 1:1,000, respectively; Santa Cruz Biotechnology, Santa Cruz, CA); Calnexin (#ADI-SPA-860-D; ENZO, Farmingdale, NY; dilution 1:1,000); α-SMA and β-actin (#5228 and #A2228; Sigma Aldrich, St. Louis, MO; dilution 1:2,000); anti-GFP (#ab6556; abcam, Cambridge, MA; dilution 1:1,000). For immunoprecipitation analysis, the following antibodies were used: Nogo-B (#AB-163; Kinasource, Scotland, UK; dilution 1:100) and anti-HA (#11815016001; Roche; dilution 1:100).
Nogo b
Manufactured by R&D Systems
Nogo-B is a laboratory product manufactured by R&D Systems. It is a protein involved in the regulation of cell growth and differentiation. The primary function of Nogo-B is to act as a negative regulator of angiogenesis, the process of new blood vessel formation.
Automatically generated - may contain errors
Lab products found in correlation
Calnexin, by Enzo Life Sciences (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Roche (1 mentions)
α sma, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Anti gfp, by Abcam (1 mentions)
S1pr1, by R&D Systems (1 mentions)
Alexa 488 anti rabbit, by Jackson ImmunoResearch (1 mentions)
Fluoview confocal microscope, by Olympus (1 mentions)
2 protocols using nogo b
1
Western Blot and Immunoprecipitation Analysis
2
Immunofluorescence Analysis of Placental Arteries
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Isolated arterial chorionic placental vessels were incubated in calcium-free Krebs for at least 30 min to allow vessel vasodilation. Subsequently, the arteries were fixed with 4% PFA and left overnight at 4 °C. PFA-fixed arteries were OCT-embedded. For immunofluorescence, frozen placental artery sections were stained for Nogo-B (1:200, R&D), S1PR1 (1:200, R&D), and CD31 (1:200, Invitrogen) overnight at 4 °C and were then stained with Cy5-labeled anti-goat antibody (#A21436, Invitrogen, 1:500) Alexa 488 anti-rabbit (#016-540-084, Jackson ImmunoResearch, 1:200) and Alexa 568 anti-mouse in PBS for 1 h. Nuclei were stained with DAPI. Confocal immunofluorescence images of the tissues were captured on an Olympus Fluoview confocal microscope and quantified with ImageJ.
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