Fig. 4.

Effect of downregulation of ERK1/2 and MEK1 on necrotic RPTC-induced P2X7 expression and cell death. NRK-49F cells were transfected with siRNA targeting ERK1 and ERK2 or scrambled siRNA and 24 h after transfection, cells were treated with necrotic RPTC for 24 h (A–D). Cell lysates were prepared and subjected to immunoblot analysis with antibodies for ERK1/2, GAPDH, P2X7, cleaved PARP, cleaved caspase-3, or α-tubulin (A and B). The expression of P2X7 was quantified by densitometry and normalized to α-tubulin level (C). Bars with different letters (a–d) are significantly different from one another (P < 0.05). NRK-49F cells were transfected with scrambled siRNA or MEK1-specific siRNA and treated with necrotic RPTC for 24 h. Then, cells were harvested and subjected to immunoblot analysis for MEK1, P2X7, or α-tubulin (E). Cell viability was determined by the MTT assay (D and F). Values are means ± SD of 3 independent experiments conducted in triplicates and expressed as the percentage of control. Representative immunoblots from 3 experiments are shown.