PTENα increases neutrophil deformability through dephosphorylation of moesin at T558. (A) In vitro dephosphorylation assay. HA-tagged moesin was purified using S-protein agarose beads (Novagen) from transfected HEK293T cells. After incubation, with or without purified PTENα, in dephosphorylation buffer at room temperature for 40 minutes, beads were washed and boiled with sodium dodecyl sulfate loading buffer. Dephosphorylation of moesin (p-moesin) was evaluated with anti–p-moesin (T558) antibody (1:5000; Abcam), and total moesin (t-moesin) was evaluated with anti-moesin antibody (1:2000; Abcam). (B) p-Moesin and t-moesin were evaluated in HeLa WT and HeLa PTENα^−/− cell lysates with anti–p-moesin (T558) and anti-moesin antibody, respectively. (C) p-Moesin and t-moesin were evaluated in HeLa PTENα^−/− cells 72 hours after transfection with tag vector, tagged PTENα, and 2 inactivated mutants (C297S and G302E). Only the PTENα-C297S mutant did not dephosphorylate moesin at T558 (lane 3). (D) Different FLAG-tagged moesin mutants and GFP-tagged PTENα were cotransfected into HEK293T cells, and cell lysates were pulled down with anti-Flag antibody and subjected to immunoblot with GFP or Flag antibody. The inactive conformation of moesin (T558A) did not effectively coimmunoprecipitate with PTENα (lane 3 vs. lanes 2 and 4). (E-F) Immunoblot analysis of p-moesin and t-moesin in isolated neutrophils (1-2 × 10⁶) Neutrophils were pelleted and lysed after stimulation with 100nM fMLP for 240 seconds or were left unstimulated. p-moesin and t-moesin were evaluated with western blot (E) and were analyzed by densitometric quantification (F) (n = 3 mice). Results are presented as mean ± standard error of the mean. ***P < .001, 2-way repeated-measures ANOVA, followed by the Bonferroni post test. ns, not significant (P > .05).