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. 2019 May 30;20(11):2653. doi: 10.3390/ijms20112653

Figure 2.

Figure 2

Inhibition of FAK activity, but not the depletion of c-Abl, reduced ERK activity in MDA-MB-231 cells, suggesting that FAK mediates CAP1 signals to regulate ERK. (A) The control and CAP1-knockdown MDA-MB-231 metastatic breast cancer cells were treated with 5 μg/mL FAK inhibitor PF-573228 (SelleckChem) for 13 h before cells were harvested for Western blotting, to detect the effects on ERK activity. Antibodies against FAK, pFAK(Tyr397), ERK, and pERK (Thr202/Tyr204) were from Cell Signaling Technology Inc., and that against GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was from Santa Cruz Biotechnology Inc. The control indicates the stable cells that harbor the empty vector of the CAP1 knockdown shRNA construct, and CAP1KD indicates CAP1-knockdown-stable cells that we previously established. (B) Transient silencing of c-Abl with the siRNA (SCBT #sc-29310) in the control and CAP1-knockdown MDA-MB-231 cells did not have a remarkable effect on ERK activity. The antibody against c-Abl was from Cell Signaling Technology Inc. Cells were cultured for 24 h in DMEM supplemented with 10% fetal bovine serum and then transfected with the siRNA, following the manufacturer’s protocol. Cells were harvested 48 h after transfection for analyses in Western blotting, to confirm the depletion of c-Abl and its effects on ERK activity.