Figure 5.

Geldanamycin reduces Hakai protein expression accompanied with an increase of Annexin A2: (a) HEK293T and HCT116 cells were treated with geldanamycin at the indicated times and concentrations. Cell lysates were collected and subjected to western blot analysis with the indicated antibodies. (b) HCT116 and HEK293T cell lines were treated with geldanamycin for 24 h at the indicated concentration. Cells were collected and subjected to mRNA extraction followed by RT-qPCR. Levels of Hakai mRNA normalized to control (HPRT) mRNA were measured and graphically represented as Mean ± SEM for three independent experiments (* p < 0.05, ** p < 0.01). (c) HEK293T cells were transiently transfected with pcDNA 3.1 (10 µg) empty vector or pcDNA-Flag-Hakai (4 µg), pBSSR-HA-ubiquitin (3 µg) and pSG-v-Src (3 µg) plasmids for 48 h. Cells were treated with 10 µM geldanamycin 24 h after transfection. Cells were collected and protein levels were evaluated by western-blotting with the indicated antibodies. (d) HCT116 cells were transfected with Hakai siRNA-1 and siRNA-2 oligos for 72 h and treated with 10 µM geldanamycin for the last 24 h of transfection. Cell lysates were collected and protein expression was evaluated by western-blotting with the indicated antibodies. Relative quantification of Annexin A2 expression levels was graphically represented as Mean ± SEM for three independent experiments (* p < 0.05, ** p < 0.01).