Anti phosphotyrosine antibody py 99

After eight weeks of treatment the animals were anesthetized using Pentobarbital (Southern Anesthesia, West Columbia, SC; 80 mg/kg). Once under anesthesia the thorax was opened and the rat perfused with saline via an injection into the heart. The kidneys were then removed and dissected free of perinephritic fat and flash frozen in liquid nitrogen. At a later time point a section of kidney (approximately 1/4 of the total kidney containing cortex tissue only) was homogenized in RIPA buffer. The homogenates were centrifuged 13,000 ×g for 10 min to clear debris and the membrane proteins and cytosol (200 μg/sample) were solubilized in 2x Laemmli buffer prior to separation by SDS-PAGE (TGF-β 4–12% gradient gel, thrombospondin-1 (TS-1) 6%, and collagen type IV 8%) followed by transfer to Immobilon P membranes. The membranes were incubated with antibodies for TGF-β, TS-1, and type IV collagen. Lysates from all of the animals in each group were analyzed. To determine if the antibody inhibited β3 phosphorylation, kidney homogenate (200 μg) was immunoprecipitated with the anti-β3 antibody (R2949) using a 1 : 500 dilution. The proteins in the immunoprecipitate were separated by SDS-PAGE. After transfer to Immobilon P β3 phosphorylation was visualized by immunoblotting with anti-phosphotyrosine antibody (pY-99, Santa Cruz) at a 1 : 1000 dilution.