FIGURE 4.

Effects of the lysosomal inhibitor, bafilomycin A1 (Baf A1), and proteasomal inhibitor, MG 132, on BK protein expressions in HEK 293 cells. HEK293 cells were transfected with BKα plasmid in combination with either control siRNA or siRNA SPAK. Sixteen hours before cell lysis, a dose of either Baf A1 (0.4 μM) or MG 132 (10 μM) was added to the culture medium. Forty-eight hours after transfection, cells were lysed and subjected to SDS-PAGE and Western blot analysis. (A) Representative immunoblots for BK, phosphorylated and total SPAK protein, and actin levels. Lanes 1–3 indicate the control siRNA group treated with Baf A1 (0.4 μM) and MG 132 (10 μM). Lanes 4–6 indicate the siRNA SPAK group treated with Baf A1 (0.4 μM) and MG 132 (10 μM). (B) Bar graph summarized from four independent experiments. Knockdown of SPAK expression (lane 4) significantly reduced total BK protein expression compared with the control group (lane 1). n = 4; ^#p < 0.05. Both Baf A1 and MG132 treatments reversed the inhibitory effects by knocking down SPAK expression (lanes 5 and 6). n = 4; *p < 0.05 compared with the untreated siRNA SPAK group (lane 4). These results indicate that the inhibitory effect of SPAK siRNA on BK protein expression is abolished by both Baf A1 and MG132.