Rabbit anti mouse gapdh

Total protein concentrations of lung homogenate lysates were determined and SDS-PAGE was performed using Mini-PROTEAN^® Precast Mini PAGE Gels (Bio-Rad, Hercules, CA). Trans-Blot Turbo Mini 0.2 µM PVDF Transfer Packs (Bio-Rad) were used for transferring of proteins to the PVDF membranes. Membranes were blocked for 3h at RT and incubated with primary antibodies (rabbit anti-human TRAP5b (provided by Dr. Göran Andersson), rabbit polyclonal to TRAP5 (Cat #PA5-116970; Invitrogen, MA, USA), and rabbit anti-mouse GAPDH (Cat #MA5-15738-D680; Invitrogen 1:500) in blocking buffer. After washing with PBS-Tween, membranes were incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti rabbit/mouse (Invitrogen, Carlsbad, CA) for 1h RT. Imaging of blots was preformed using a ChemiDoc system (Bio-Rad) followed by quantification with densitometry normalized to GAPDH.

To evaluate the levels of IL-1β and CD68 in the aortic wall of the Ang-II + 01BSUR group and Ang-II group, Western immunoblot analysis was performed. Aortic tissues (n = 3 per group) were homogenized and solubilized in RIPA buffer (pH = 7.4). The protein concentration was determined using UV-spectrometry at 280 nm (Evolution 220 UV-Visible Spectrometer, Thermo Scientific^TM). 5 mg/ml total protein per lane diluted in SDS loading buffer was loaded onto 4-12% Tris-Glycine Gels (ServaGel TG Prime Vertical Tris-Glycine Gel 4-12%). Proteins were separated by SDS-PAGE (Hoefer SE260). To assure equal amounts of total protein loaded per lane, GAPDH was used as loading control. Due to the identical molecular weights of the targeted proteins CD68 and GAPDH, 2 gels were run simultaneously. Proteins were transferred to PVDF membranes using Trans-Blot Turbo RTA transfer kit (Bio-Rad). Primary antibodies (Rabbit anti-Mouse IL-1β, Bio-Rad 1:500; Rabbit anti-Mouse CD68, antibodies.com, 1:500; Rabbit anti-Mouse GAPDH, Invitrogen 1:1000) were detected using a secondary HRP-labeled antibody (Goat anti-Rabbit IgG (H/L): HRP, Bio-Rad 1:1000) and SeramunBlau® blotting substrate (Seramun Diagnostica GmbH). Protein bands were quantified by ImageJ (Version 1.51).