Expressplus page gel 8 16

Whole-cell BMDM protein lysate was isolated using RIPA buffer supplemented with protease inhibitors (Roche, 11873580001) and protein concentration determined by BCA assay.
Pre-cast gels (ExpressPlus PAGE gel 8–16%, genscript, M81612) were used to and proteins were transferred to a nitrocellulose membrane. Primary antibodies directed against HIF1α (Novus Biologicals, NB100-499), HIF2α (Novus Biologicals, NB100-122), and β-Actin (Abcam, ab8227), were followed by appropriate HRP-labelled secondary antibody incubation. Signal was developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher scientific, 34095) and visualized using a digital scanner. Band density was quantified with ImageJ, and normalized for total proteins by β-actin as loading control.

Human carotid plaques were snap-frozen immediately after collection. Sample preparation procedures were carried out on dry ice. Carotid plaques were divided in smaller pieces (± 0.5 cm) and manually grinded under constant addition of liquid nitrogen. The resulting tissue dust was incubated with 500 µl TRIS lysis buffer and EDTA-free protease inhibitor cocktail (Roche, 04693159001). Subsequently, protein liberation was further enhanced by crushing grinded tissue in a Beadbeater, and sonicating the sample for 2 min. Upon centrifugation (maximal speed, 5 min) supernatant was collected and protein concentration was determined using BCA kit (Pierce, Cat. No. 23227). Pre-cast gels (ExpressPlus PAGE gel 8–16%, genscript, M81612) were used for protein seperation, and transferred to a nitrocellulose membrane. CAIX was detected using primary antibody M75 (1:3000) followed by HRP-labeled secondary antibody incubation (Jackson, 715-035-150). Signal was developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher scientific, 34095) and visualized using a digital scanner. Signal intensity was quantified using ImageJ Gel Analyzer software and normalized for total protein content using intensity of Ponceau S. Each gel contained the same plaque sample, allowing normalization between gels.