Figure 1.

The importance of alkylating the β-tubulin Cys³⁵⁴ residue and CCT-β expression in I-Trp-induced cell apoptosis. (a) Interrupting PPI of β-tubulin:CCT-β by I-Trp leads to cell apoptosis. (b) The expression levels of β-tubulin, CCT-β, green fluorescent protein (GFP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the HEK-293 cell lysates expressing the pIRES2-EGFP plasmid DNA either without (mock) or with wild-type β-tubulin (β-tub/wt) or C354S mutant (β-tub/C354S) were assessed by immunoblotting with the corresponding antibodies. (c) Incorporation of I-Trp into β-tubulin Cys³⁵⁴ is required for eliciting apoptosis of HEK-293 cells. The cells were treated with I-Trp at the indicated concentrations. Apoptosis was measured 24 h after treatment. (d) The expression levels of CCT-β, β-tubulin and GAPDH were analyzed by western blotting using specific antibodies for the HEK-293 cell lysates either untreated or treated with stably expressing control (CTL) or CCT-β shRNA. (e) Knockdown of CCT-β inhibits I-Trp-induced apoptosis of HEK-293 cells. Cells were treated with I-Trp at 5 μM. Apoptosis was assessed 24 h after treatment. GFP was used as CTL for the transfection procedure. GAPDH was used as protein loading CTL. The results in (c) and (e) represent the mean of three independent experiments. The values shown represent the mean±S.D. The symbol ‘***' denotes statistical significance at P<0.001. NS represents results that are not significant