Figure 5

PMA fails to overcome ApxI-induced reduction in FAK and Akt activity and cytotoxicity. (a–c) Porcine AMs were treated with 200 nM PMA or 0.1% DMSO (Vehicle) for 0–60 min. Cell lysates were subjected to (a) Western blot analyses for p-FAK^Y397 and p-Akt^S473. The grouping of blots was cropped from different portions of two gels and exposed separately. Black lines delineate the boundary between not contiguous lanes of the same gel. Uncropped blot images for (a) are presented in Supplementary Figure S4. The average intensities of (b) p-FAK^Tyr397 and (c) p-Akt^Ser473 are from three independent experiments and normalized to the intensity of β-actin. Asterisks indicate significant differences compared to the 0-min treatment group. *p < 0.05. (d–f) Porcine AMs pretreated with 200 nM PMA for 15 min were stimulated with 0 or 2.5 CU/ml of ApxI for 0–5 min. Cell lysates were collected and subjected to (d) Western blot analyses for p-FAK^Y397, p-Akt^S473, and β-actin. The grouping of blots was cropped from different portions of two gels and exposed separately. Uncropped blot images for (d) are presented in Supplementary Figure S5. The average intensities of (e) p-FAK^Tyr397 and (f) p-Akt^Ser473 are from three independent experiments and normalized to the intensity of β-actin. Asterisks indicate significant differences compared to the non-ApxI treatment group at the identical time point. *p < 0.05; **p < 0.01. (g) Porcine AMs were incubated with 200 nM PMA or 0.1% DMSO (Vehicle) for 15 min, then incubated with 0–10 CU/ml of ApxI for 90 min. The percent cytotoxicity was quantified using the LDH release assay. Data are from three independent experiments of triplicate determinants. *p < 0.05.