Figure 9.

AT1002 increases ZO-1 serine/threonine phosphorylation. A) Postconfluent IEC6 monolayers were treated for 30 min with increasing concentrations of AT1002 or medium alone and lysed. ZO-1 was immunoprecipitated (IP) from 500 μg of total cell lysate; ZO-1 immunoprecipitates were processed for phosphoserine immunoblotting (IB) and then were stripped and reprobed for phosphothreonine immunoblotting (IB*). To ensure equal loading and transfer, immunoblots were stripped and reprobed with immunoprecipitating anti-ZO-1 antibody (IB**). B) IEC6 monolayers were treated for increasing times with AT1002 (100 μM) or medium alone and lysed. ZO-1 was immunoprecipitated from 500 μg of total cell lysate, and the ZO-1 immunoprecipitates were processed for phosphoserine immunoblotting (IB) and then were stripped and reprobed for phosphothreonine immunoblotting (IB*). To ensure equal loading and transfer, the immunoblots were stripped and reprobed with the immunoprecipitating anti-ZO-1 antibody (IB**). Molecular mass (kDa) is indicated at left. Blots are representative of ≥3 independent experiments. C, D) For the phosphoserine (C) and phosphothreonine blots (D), densitometric quantification of each phosphoserine and phosphothreonine signal was normalized to the ZO-1 signal for the same lane on the same stripped and reprobed blot. Vertical bars represent mean ± se fold change in arbitrary densitometry units of phosphoserine/threonine signal normalized to arbitrary densitometry units of ZO-1 signal, each relative to the simultaneous control (n=4). *P < 0.05 vs. medium control.