Fig. 1. MAPK signaling induced by T. gondii requires host Ca²⁺. (A) Fura-2-loaded macrophages were incubated in normal buffer (N) or BAPTA-AM (15 µM) in Ca²⁺-free EGTA medium (E/B) as described in Materials and Methods, then transferred to the Ca²⁺ imaging chamber, and stimulated with thapsigargin (thg; 1 µM, arrowhead) in normal buffer or Ca²⁺-free EGTA buffer, respectively, to release available intracellular Ca²⁺ stores. Images were captured at 5 frames per minute and 340/380 ratios were calculated and averaged to create the line tracings depicted. Averages are based on ratios collected from approximately 60 cells in each imaging field. (B-D) Macrophages were pre-treated with normal medium (N) or BAPTA-AM (15 µM) in Ca²⁺-free EGTA medium (E/B) for 45 minutes, washed, then treated with medium (m) as a control or infected with live T. gondii at a ratio of 5:1. Whole cell lysates were collected at the times indicated, and subjected to immunoblotting for phospho-ERK1/2 and phospho-p38 using individual antibodies specific to each MAPK (B, top panels). Membranes were then stripped and reprobed for total levels of ERK1/2 and p38 (bottom panels). Separate lysates were collected and used to immunoblot for phospho-MEK1/2 (C) or phospho-STAT3 (D). The membranes were stripped and reprobed for ß-actin to determine loading. Each experiment was repeated two to four times, and representative results are presented.