Figure 4.

PTP inhibition abrogates the Cr(VI)-induced decrease in Akt expression and activity. (A) HLF cells were treated with and without 10 µM SOV for 1 h. Total protein was extracted and immunoblotting for PTEN from the pan-pTyrosine immunoprecipitates and for pTyrosine from the PTEN immnuoprecipitates was performed. Non specific IgG was used as the negative control. (B) HLF cells were treated with 0, 1, 3 or 6 µM Cr in the presence and absence of 10 µM SOV for 24 h. Total cellular protein was extracted after the treatment. Proteins were separated by SDS-PAGE and total pSer 473 Akt was detected by immunoblotting. The same amount of protein loading was confirmed by immunoblotting for β-actin. Data are the means ± SE of three independent experiments and are expressed as percentage of control, normalized to β-actin. * indicates a statistically significant difference from control (p < 0.05) and ⁺ indicates a statistically significant difference between the samples treated with and without SOV (p < 0.05). (C) HLF cells were treated with 0, 1, 3 or 6 µM Cr in the presence and absence of 10 µM SOV for 24 h. Total cellular protein was extracted, and Akt was immunoprecipitated and used in an in vitro kinase assay with a Gsk-3 fusion protein as a substrate. Proteins from each reaction were separated by SDS-PAGE, and Gsk-3 phosphorylation was detected by immunoblotting, and normalized to total Gsk-3 protein expression. Data are the means ± SE of two independent experiments are expressed as percentage of control, normalized to total GSK3 and + indicates a statistically significant difference between samples treated with and without SOV (p < 0.05). Cr(VI) induced a statistically significant (p < 0.05) decrease in Akt in vitro activity as determined by linear regression analysis (r² = 0.9647).