Sc 21712

The cell extract samples were made according to the methodology described (Shoshani, 2004 (link)) and were prepared with the same amount of protein for SDS-PAGE electrophoresis on a Bio-Rad miniProtean III apparatus (Bio-Rad, Hercules, CA, USA) and then the proteins that ran in the gel were transferred to nitrocellulose membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The Ponceau red method was used to ensure equal loading protein for all samples. The membrane was then blocked for 1 h, washed with Tris-buffered saline plus 0.1% Tween-20 (T-TBS) and incubated overnight with monoclonal antibodies specific for α1-Na/K-ATPase (sc-21712, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PMCA4 antibody (JA9 - MA1-914, Thermo Fisher), β-actina (sc81178, Santa Cruz Biotechnology). The next day the membrane was washed with T-TBS and incubated for 2 h with peroxidase enzyme conjugated secondary antibody (sc-516102, Santa Cruz Biotechnology). The membrane was developed using an equimolar mixture of two solutions (Solution 1: Tris 100 mM pH 8.5, 2.5 mM Luminol and 0.396 mM p-coumaric acid; Solution 2: 100 mM Tris pH 8.5 and 0.06% H₂O₂). Immunoblots were quantified by densitometry using the ImageJ software (http://rsb.info.nih.gov/ij). Several exposure times were analyzed to ensure the linearity of the band intensities.