FIGURE 1:

JAM-A is tyrosine phosphorylated at Y280. (a) Human JAM-A highlighting tyrosine 280 (murine JAM-A Y281) within the cytoplasmic tail in close proximity to S284. (b) Using a commercially available phosphospecific antibody, phosphorylation of JAM-A Y280 (p-Y280) is undetectable in confluent monolayers of SK CO-15 cells under basal conditions, but robust phosphorylation is observed after global inhibition of tyrosine phosphatase with pervanadate (25 μM) for 60 min (left). Detection of p-JAM-A Y280 by immunoblot after pervanadate treatment (60 min), compared with nontreated controls. Treatment with calf intestinal phosphatase (CIP) reversed pervanadate-induced phosphorylation of JAM-A Y280 (right). Similar results were observed in T84 cells. (c) Confluent SK CO-15 cells were treated with pervanadate as time points indicated, then cells were fixed and costained with anti–p-JAM-A Y280 and total JAM-A antibodies. (d) T84 cells were grown on Transwell filters until confluent. After 45 min of pervanadate treatment, cells were fixed with 4% PFA and permeabilized with 1% SDS followed by immunostaining with p-JAM-A Y280 and total JAM-A. Confocal Z-stacks indicate p-JAM-A Y280 localizes to the TJ. We also detected p-JAM-A Y280 localization to TJ in SK CO-15 cells (unpublished data). Scale bar: 10 μm.