Serine phosphorylation increases K6a solubility. (A) Cytosolic K6a was immunoprecipitated from hTCEpi organotypic culture treated with various bacterial ligands followed by LC-MS analysis. Four different phosphopeptides were identified. (B–D) The degree of modification (abundance of phosphoform/abundance of unmodified form) was determined for each phosphopeptide. The fold amount of each modification after treatment relative to unstimulated control (basal levels = 1) is presented. (E) Phosphorylation of K6a positively correlates with its cytosolic level in hTCEpi organotypic culture. hTCEpi cells were treated with DMSO or 200 nM phosphatase inhibitor calyculin A for 5 h before they were harvested and immunoprecipitated (IP) with preimmune serum or anti-K6a antiserum. Samples were immunoblotted (IB) by anti-K6a antiserum or antiphosphoserine antibody. (F) Dephosphorylation of K6a by CIP. Remaining eluates from calyculin A–treated samples from E were divided into three fractions, resolved, transferred to polyvinylidene difluoride membrane, and incubated with CIP (fraction 3), without CIP (fraction 2), or CIP buffer only (fraction 1). Membranes were immunoblotted for K6a (fraction 1) or phosphorylated K6a (fractions 2 and 3).