Lnk inhibits cell growth and signaling in HEL cells. (A-B) HEL cells (5 × 105) expressing different Lnk forms or WT APS were cultured in complete media, and viable cell numbers were determined by trypan blue exclusion at the indicated times with 4 weeks interval (B). Data represent the mean counts ± SD (error bars) of viable cells from triplicate determinations and are representative of at least 3 independent experiments. GFP expression level in all clones was determined by fluorescence-activated cell sorting; (C) WT and R364M Lnk forms and APS protein expression was assessed with anti-Lnk and anti-APS antibodies, respectively. Actin blot is shown as equal loading control. Vector, ♦; R364M, □; APS, ●; Lnk, Δ. (D) Total lysates from HEL cells expressing different Lnk forms or WT APS were prepared as in Figure 4B at day 4 (left panels) and day 35 (right panels) of culture. JAK2, STAT5, and MAPK activation was assessed by immunoblotting with anti-phospho-JAK2 (p-JAK2), anti-phospho-STAT5 (p-STAT5) or anti-phospho-Erk1/2 (p-Erk) antibodies, respectively. Total JAK2, STAT5, and Erk levels were analyzed by immunoblotting. White space gaps indicate repositioned gel lanes.
