Imagequant tl software v8

Protein concentrations of whole kidney samples were determined using the BCA protein assay kit (Thermo Fisher, Belgium) . Samples containing the same amount of proteins were separated on a 12% SDS-page gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Belgium), then transferred to a polyvinylidene fluoride membrane and blocked 2h with 5% bovine serum albumin (BSA) in Tris-buffered solution containing 0.1% Tween-20 (TBS-T). To investigate whole kidney RAAS protein expression, the membrane was incubated overnight at 4°C in the presence of an antiangiotensin II type I receptor antibody (ATIIT1R, 1/2000, ab18801, Abcam, UK), as previously described (Wang et al., 2015) . Secondary swine anti-rabbit horseradish peroxidase-conjugated antibody (P0217, DAKO, Belgium) at a dilution of 1/2500 was used. Both primary and secondary antibodies were diluted in BSA-TBS-T. ATIIT1R was visualized using the chemiluminescence (ECL) technique (1 min exposure) using the Pierce ECL Plus Western Blotting Substrate Kit (Thermo Fisher, Belgium) and quantified using Image Quant TL software v8.1 (GE Healthcare Europe, Belgium). Data were normalized to β-actin protein levels (1/2500, sc-4778, Santa Cruz, USA) (Cops et al., 2018a; Cops et al., 2018b ) (figure S1).