Calcium responses are altered in Pak1^−/− BMMCs. IgE-primed WT and Pak1^−/− BMMCs were loaded with Ca²⁺-sensitive dye (fura2) and suspended in Ca²⁺-containing medium (A). To detect release from intracellular stores, cells were suspended in Ca²⁺-free media and treated with EGTA prior to stimulation (B). FcϵRI was activated by addition of DNP-HSA and changes in intracellular calcium concentration (i[Ca²⁺]) were measured by spectrophotofluorimetry. (A) Representative experiment of a WT (black) versus Pak1^−/− (red) BMMC sample demonstrating decreased Ca²⁺-bound fura-2 in the Pak1^−/− cells after antigen stimulation (left panel). Changes in i[Ca] after addition of digitonin followed by EGTA are also shown. Average change in i[Ca²⁺] (right panel) expressed as a percentage of WT change in i[Ca²⁺] from 4 independent experiments, *P < .05, WT versus Pak1^−/−, unpaired, 2-tailed, Student t test. (B) Representative experiment of a WT (black) versus Pak1^−/− (red) BMMC sample demonstrating similar responses upon antigen stimulation after depletion of extracellular calcium (left panel). Average percentage increase in i[Ca²⁺] (right panel) from baseline in WT and Pak1^−/− BMMCs from 3 independent experiments, P < .0.57, WT versus Pak1^−/−, unpaired, 2-tailed, Student t test. (C) Western blot and densitometry for activated PLCγ1. NS indicates cells at baseline (nonprimed, nonstimulated). IgE-primed WT and Pak1^−/− BMMCs were stimulated with DNP for 30 seconds or 1 minute. Cell lysates (“Western blotting”) were subjected to immunoblotting with anti–phospho-PLCγ1 (top blot) or anti–total PLCγ1 (bottom blot) and densitometry was performed. Intensity of phosphorylated PLCγ1 bands is represented as the ratio of phospho/total PLCγ1 for 1 of n = 4 experiments (P = .79 and P = .47 at 30 seconds and 60 seconds, respectively).