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. 2013 Dec;15(12):1379–1390. doi: 10.1593/neo.131434

Figure 1.

Figure 1

BCA2 inhibits AMPKα1 phosphorylation and activation. (A) The assessment of basal protein expression levels in the four indicated human breast cancer cell lines. Exponentially growing cells were lysed for Western blot analysis with specific antibodies to BCA2, pAMPK (Thr172), total AMPKα1, pAKT (S473), total AKT, and β-actin (loading control). (B) Immunoblot of MDA MB 231 cells (grown in a six-well plate) transfected with BCA2 siRNA (x-axis, µg) for 72 hours. Scrambled, non-silencing siRNA and untreated cells were used as controls. Cell extracts were used for Western blots with specific antibodies to those in A as well as pACC (Ser79) and total ACC. Densitometry analysis is a representative of the mean ± SEM for three independent experiments. pAMPK was normalized to total AMPKα, pACC to total ACC, and BCA2 to β-actin. Fold change was calculated compared to the control. (C, D) MCF7 (C) and MDA MB 468 (D) cells were transfected with 5 µg of either BCA2 siRNA or scrambled control for assessment of the effect on pAMPK as done in B. (E) Immunoblot of HEK293T cells co-transfected with BCA2 wt and AMPKα1 (2.5 µg each). After a 48-hour transfection, cells were either treated with metformin (20 mM) or AICAR (1 mM) for 6 hours or left untreated. Cell extracts were used for Western blots with specific antibodies as previously stated. FLAG was used as a measure of transfection efficiency.