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. 2006 Apr;168(4):1107–1118. doi: 10.2353/ajpath.2006.050959

Figure 3.

Figure 3

Effect of MBCD on basal and EGF-induced Akt activation. A: Serum-starved A431 cells were treated with 1, 5, or 10 mmol/L MBCD for 1 or 2 hours or with 30 nmol/L EGF for 5 minutes, followed by cell lysis with 2× sample buffer. Aliquots (30 μg of protein) from each treatment were subjected to immunoblotting analysis using anti-phospho-EGF receptor antibodies that specifically recognize tyrosine-phosphorylated EGF receptors at 845 or 1068 residues. B: Serum-starved A431 cells were treated with 5 mmol/L MBCD for 0 to 6 hours or with 30 nmol/L EGF for 5 minutes, and cells were lysed with 2× sample buffer. Thirty micrograms of protein from each treatment was subjected to immunoblotting analysis using antibodies specific for phospho-Akt, Bcl-xL, caspase-3, PARP, active-ERK1/2, and ERK1/2 (loading control). C: Serum-starved A431 cells were pretreated with 5 mmol/L MBCD and then washed with medium, followed by 30 nmol/L EGF treatment for 10 minutes. Thirty micrograms of protein from each treatment was subjected to immunoblotting analysis using anti-phospho Akt and anti-total Akt antibodies. D: Serum-starved A431 cells were pretreated with 5 mmol/L MBCD for 1 or 2 hours and then washed with medium, followed by 30 nmol/L EGF treatment for 2 hours. Cell viability was measured by MTS assay. Values represent the means ± SD of three independent experiments. These experiments were performed two separate times with comparable results.