Fig. 4. Pervanadate induces kAE1 phosphorylation on residues Y359 and Y904 and is sensitive to Src kinase family inhibitors. (A,B) Western blots of Bric170 immunoprecipitates from MDCKI or MDCKI-kAE1 cells treated with or without pervanadate for the indicated times. The membranes were sequentially probed with anti-Y904-P, anti-AE1Ct and anti-Y359-P, stripping after each antibody application. (A) kAE1 phosphorylation on Y359 and Y904 is detectable by the phosphospecific antibodies within 1 minute and the majority of the protein is phosphorylated within 5 minutes. The blot shown is representative of at least eight similar experiments. (B) MDCKI-kAE1 cells were preincubated with Src kinase inhibitors 25 µM PP1, 25 µM PP2, 25 µM PP3 (inactive analogue), 2 µM staurosporine (Stauro), 100 µM genistein (Gen), 5 µM herbimycin (Herb) and 25 µM SU6656 for 30 minutes before addition of 200 µM pervanadate for 5 minutes. kAE1 phosphorylation on residues Y359 and Y904 was inhibited by both PP1 and PP2, and to a lesser extent by staurosporine and SU6656. The broad-spectrum Src kinase inhibitors genistein and herbimycin did not inhibit kAE1 phosphorylation under the conditions tested. The blot shown is representative of at least three independent experiments for each inhibitor.