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. 2021 Oct 27;66(2):223–234. doi: 10.1165/rcmb.2021-0192OC

Figure 4.

Figure 4.

Overexpression of Plk1 enhances Casp9 phosphorylation at Ser-196, but not Thr-125 in HASM cells. (A) Representative IBs showing the effects of Plk1 overexpression on Casp9 phosphorylation at Ser-196/Thr-125, Casp9/3 expression and ERK1/2 phosphorylation. HASM cells were transfected with Ctrl plasmids or plasmids encoding Plk1 for 24 hours and serum starved for additional 24 hours. They were then treated with 10 ng/ml PDGF-BB for 24 hours in the presence or absence of 10 μM U0126. IB analysis was used to evaluate protein phosphorylation and expression. (B) Protein ratios of Plk1/GAPDH are normalized to Ctrl cells not treated with PDGF-BB or U0126. Plk1 overexpression is verified in these cells. (C–G) Casp9 phosphorylation at Ser-196 (C), pro-Casp9/3 (D and E), ERK1/2 phosphorylation (ERK1: Thr-202/Tyr-204; ERK2: Thr-185/Tyr-187) (F), and Casp9 phosphorylation at Thr-125 are normalized to Ctrl cells not treated with PDGF-BB or U0126 (G). (H) Apoptosis of HASM cells overexpressing Plk1 was evaluated using the Annexin V apoptosis assay kit. RLU = relative luciferase unit. Data are mean values of five to six nonasthmatic cell cultures from three donors. Error bars indicate SD. *P < 0.05 and **P < 0.01. Two-way ANOVA was used for statistical analysis.