Quantitative Western Blot Analysis of Aorta and PVAT

Western blot analysis was performed with lysates of aorta and PVAT as described (Conklin et al., 2015 (link); Haberzettl, McCracken, et al., 2016 (link)). Briefly, collected tissues were lysed in RIPA buffer (50 mM Tris•HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 0.25% sodium desoxycholate; 1% NP‐40; 1:100 protease inhibitor cocktail, Pierce, Rockford, IL; 1:100 phosphatase inhibitor, Sigma‐Aldrich) and proteins were separated by SDS‐PAGE and transferred to PVDF membranes (Bio‐Rad, Hercules, CA). Membranes were probed with antibodies against phospho‐Akt (Ser473) and Akt (1:1000; Cell Signaling Technology, Danvers, MA), or protein–acrolein adducts, and then developed using ECL® plus reagent (Amersham Biosciences, Piscataway, NJ). Detected band intensities (Typhoon 9400 variable mode imager, Amersham Biosciences) were quantified using Image Quant TL software (Amersham Biosciences) and normalized either to appropriate loading controls or total protein staining (amido black).