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. 2013 Aug 28;8(8):e73296. doi: 10.1371/journal.pone.0073296

Figure 2. CAR shedding is stimulated by the calcium ionophore ionomycin and the phorbol ester PMA.

Figure 2

(A) U87 CAR cells were treated with the calcium ionophore ionomycin at the indicated concentrations for 30 minutes. Ionomycin treatment stimulated the ECD shedding of CAR. (B) PMA treatment (25 ng/ml) of U87 CAR cells did not trigger CAR ECD shedding within one hour. At this concentration, 4 hours of PMA treatment led to robust CAR ECD shedding, but remained lower than constitutive shedding over 16 hours. Volumes of conditioned media loaded on SDS-PAGE were adjusted according to lysate protein concentrations. (C) The protein kinase C (PKC) inhibitor Gö 6983 decreased PMA-stimulated shedding of CAR ECD into conditioned media in a dose-dependent manner. PMA was used at a final concentration of 1 μM, and Gö 6983 at the following concentrations: + = 1 nM; ++ = 10 nM, and +++ = 100 nM. For the Western blots shown in these panels, the anti-CAR N-terminus antibodies 2239 or 2240 were used.