Vascular Signaling Pathway Analysis

Aortic inflammation and vascular insulin and VEGF signaling were analyzed by performing Western blots to examine IκBα degradation, a marker of NF-κBα activation, and insulin or VEGF-induced Akt phosphorylation (17 (link)–20 (link), 31 (link)). In addition, IκBα degradation was examined to test for the activation of the NF-κBα pathway by FFA in HUVECs. As described before (17 (link)–20 (link), 30 (link), 31 (link)), tissue and cells were lysed, and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, Hercules, CA), and probed with antibodies against IκBα and actin or phospho-Akt (Ser473) and Akt. Antibodies against IκBα, phospho-Akt (Ser473), and Akt (1:1,000) were obtained from Cell Signaling Technology (Danvers, MA), and the actin (1:2,000) antibody was purchased from Sigma-Aldrich. The blots were developed with ECL plus (Amersham Biosciences, Piscataway, NJ; or Pierce, Thermo Fisher, Waltham, MA), and band intensities (Typhoon 9400 variable mode imager Amersham Biosciences; or myECL Imager, Thermo Fisher) were quantified using Image Quant TL software (Amersham Biosciences, Piscataway, NJ; or myImageAnalysis Software, Thermo Fisher).

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Hill B.G., Rood B., Ribble A, & Haberzettl P. (2021). Fine particulate matter (PM2.5) inhalation-induced alterations in the plasma lipidome as promoters of vascular inflammation and insulin resistance. American Journal of Physiology - Heart and Circulatory Physiology, 320(5), H1836-H1850.

Publication 2021

PVDF membranes ECL plus Typhoon 9400 variable mode imager myECL Imager Image Quant TL software myImageAnalysis Software

Actin Antibodies Antibody Aortic Cells Inflammation Insulin Membranes Phosphorylation Proteins Pvdf Sds page Tissue Typhoon Vascular Vegf Western blots

Corresponding Organization : University of Louisville

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Variable analysis

independent variables

FFA (free fatty acids)

dependent variables

IκBα degradation (a marker of NF-κBα activation)
Akt phosphorylation (induced by insulin or VEGF)

control variables

Protein separation by SDS-PAGE
Protein transfer to PVDF membranes
Antibodies used for probing (IκBα, phospho-Akt (Ser473), Akt, actin)
Imaging methods (ECL plus development, band intensity quantification using imaging software)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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