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Figure 1. Ca2+ entry through CRAC channels activates cPLA2 and LTC4 secretion through the ERK pathway. Aa) Western blots show that stimulation with 2–5 µM thapsigargin (4 min) results in the phosphorylation of ERK1/2 (denoted in upper panel as ERK1/2-P), indicative of ERK activation. Phosphorylation is prevented by removing external Ca2+. The last lane shows that the MEK1/2 inhibitor U0126 (20 µM pretreatment for 15 min) suppresses thapsigargin-induced ERK phosphorylation. Total ERK2 levels are shown (lower panel), as an indicator of constant loading of protein into each lane. Ab) Western blot showing thapsigargin-evoked ERK activation is prevented by the CRAC channel blocker 2-APB (30 µM). Loading control is shown in lower panel. Ac) Mitochondrial depolarization with either FCCP (5 µM) or rotenone (1 µg/ml) reduces ERK phosphorylation (both applied in the presence of 1 µg/ml oligomycin). Oligomycin alone had only a weak effect. Loading control is shown in the lower panel. B) Thapsigargin stimulates phosphorylation of cPLA2 on Ser-505. Upper panel shows a Western blot with a monoclonal antibody (mAb) that specifically recognizes Ser-505 cPLA2. The lower panel plots quantitative analysis from three such gels. Thapsigargin induced a significant increase in Ser-505 phosphorylation compared with control (P<0.01), and the increase was substantially reduced by either U0126 or PD98059 (P<0.01 for both cases). C) U0126 and PD98059 inhibit arachidonic acid release following CRAC channel opening. D) U0126 inhibits LTC4 secretion in a concentration-dependent manner. E) Cytoplasmic Ca2+ signals arising from store-operated Ca2+ entry are unaffected by U0126 (20 µM; 15 min pretreatment; 33 cells for control and 37 cells for U0126). Cells were pretreated with thapsigargin in Ca2+-free solution for 10 min before external Ca2+ was readmitted. F) The amplitude of ICRAC was not affected by U0126 (5 cells for each condition). *Denotes P < 0.01.
