Hrp conjugated anti rabbit immunoglobulin

Cells were lysed in Laemmli Sample buffer (Bio‐Rad, Hercules, CA, USA) containing DTT and 1% protease inhibitor cocktail. Nuclear extracts at 24 h after TGF‐β (R&D Systems, Minneapolis, MN, USA) treatment or at 48 h after TGF‐β receptor inhibitor (A‐83‐01, Wako, Tokyo, Japan) treatment were prepared by using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). The samples were separated using 12% SDS‐PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% membrane blocking agent (GE Healthcare, Buckinghamshire, UK) in PBS, and probed with anti‐SIX1 antibody (1:250, Acris Antibodies GmbH, Herford, Germany) or anti‐phospho‐smad2/3 antibody (1:500, Cell Signaling Technology, Danver, MA, USA) at 4°C overnight, anti‐α tubulin antibody (1:1000; Santa Cruz, Dallas, Tx, USA) at room temperature for 1 h or anti‐β actin antibody (1:2000; Cell Signaling Technology) at room temperature for 2 h, then washed and incubated with HRP‐conjugated anti‐rabbit immunoglobulin (DAKO) at room temperature for 2 h. Immunoreactive protein bands were identified with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).

Homogenates of whole kidneys were obtained and glomerular and tubular fractions were separated by graded sieving as described previously.21 (link),24 (link) Homogenate proteins (20 μg) were loaded on 4/20% gels, separated by electrophoresis, the protein bands transferred to polyvinylidene difluoride membranes (Millipore Co, Billerica, MA, USA), incubated with rabbit antihuman prorenin antibody (1:250 dilution), rabbit anti-PRR antibody (1:500 dilution), or rabbit antihuman angiotensinogen antibody (1:500 dilution) as the primary antibody, and with HRP-conjugated antirabbit immunoglobulin (Dako) as the secondary antibody (1:1,000 dilution). The blots were detected by DAB with nickel chloride. As loading control, the membranes were incubated with anti-β-actin antibody (Abcam, Tokyo, Japan), and processed as mentioned earlier. The area and density of bands for each protein were measured using a NIH image software program. The specificity of the antibody for human angiotensinogen was confirmed by Western blot analysis using increasing amount (10, 100, 1,000 ng) of rat angiotensinogen and human angiotensinogen on the membrane.