Differential protein expression and phosphorylation of HCK in AHI-1–suppressed and –overexpressed CTCL cells. (A) Up-regulation of 2 isoforms of HCK (p61 and p59) was evident in AHI-1–suppressed Hut 78/sh4 cells compared with the control Hut 78 cells. Reduced HCK protein expression and phosphorylation were observed when AHI-1 was overexpressed in Hut 78 cells. In addition, an uncharacterized 85-kDa band was also observed. (B) Quantification of the differential expression and phosphorylation of HCK for both the p61^HCK and p59^HCK isoforms relative to actin. (C) Peptide competition experiment in which the HCK primary antibody was incubated with 5-fold of its cognate epitope before use in the Western blot. Dissipation of the doublet bands (p61 and p59) and the uncharacterized 85-kDa band indicated that these results were specific for the HCK antibody. (D) Additional peptide competition experiment in which the HCK antibody was incubated with 5-fold of a random peptide (AHI-1) to show that the previous competition experiment was a direct result of saturation of the HCK antibody. Values shown are the mean ± SEM. The P values presented are the result of 2-sample t tests comparing the expression in cells with AHI-1 suppression with that of Hut 78 controls.