Regulation of PKC Mediated Signaling by Calcium during Visceral Leishmaniasis
Figure 3
Effects of EGTA and ionomycin on mRNA and protein expression of PKC-β, PKC-ζ, SERCA3 and PMCA4 in Leishmania-infected macrophages.
Macrophages (2×106 cells) were infected with L. donovani (1∶10) and with 2 mM EGTA and/or 1 µM Ionomycin respectively. After 4 hr, cells were washed and incubated for another 3 hr at 37°C. The cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. PKC-β, PKC-ζ, SERCA3, PMCA4, and GAPDH PCR products were resolved on an agarose gel (1.5%) and quantified densitometrically using lab software as described in Methods (A). In a similar experiment, cell lysates were prepared from EGTA and/or ionomycin treated infected macrophages, followed by Western blot for PKC-β, PKC-ζ, SERCA3, PMCA4, and GAPDH (B). Data presented above (A and B) is a representative one of three experiments conducted under identical conditions.