BAG3 Protein Is Involved in Endothelial Cell Response to Phenethyl Isothiocyanate

Figure 2

PEITC induces Rac1 activity via PI3K, and Rac1 in turn activates JNK. (a) HUVECs were treated with 0.01% DMSO (C) or treated with 10 μM of PEITC (P) for 2 h and subjected to Western blot analysis of total proteins to measure the effect of PEITC on Rac1-GTPase activity and Rac1 total protein levels and on JNK activation. The left and middle panels display columns representing densitometric analysis of Rac1 activity and of Rac1 total protein levels (expressed as Rac1/GAPDH ratios). Representative blot analysis for Rac1 after a pull-down assay and for total Rac1 protein. (b) HUVECs were treated as described above. Total cell protein content was subjected to Western blot analysis to measure phospho-JNK levels. The left panel displays columns representing densitometric analysis of the data (expressed as phospho-JNK/JNK ratios) corresponding to the right panel. The anti-GAPDH antibody was used as an internal loading control. (c) The levels of GTP-bound Rac1, total Rac1, and phospho-JNK in lysates of pretreated or not with 10 μM of LY2940021 (1 h) were analyzed in HUVECs in the presence or absence of 10 μM PEITC stimulation for 2 h. The lower bar graphs depict densitometric analysis of Rac1 activity, Rac1 protein levels, and phospho-JNK levels. (d) The levels of GTP-bound Rac1, phospho-Akt, and phospho-JNK were analyzed in lysates of pretreated or not with 100 μM of NSC23766 HUVECs for 4 h, in the presence/absence of 10 μM PEITC stimulation for 2 h. The lower bar graphs depict densitometric analysis of Rac1 activity, phospho-JNK, and phospho-Akt levels. Results were obtained from at least two independent experiments and are expressed as the mean ± SEM. , , and , statistically significant differences, compared to DMSO-treated cells (C), were calculated by Student’s t-test for unpaired data.

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