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Figure 6 | Breast Cancer Research

Figure 6

From: Insulin-like growth factor 1 attenuates antiestrogen- and antiprogestin-induced apoptosis in ER+ breast cancer cells by MEK1 regulation of the BH3-only pro-apoptotic protein Bim

Figure 6

Blockade of MEK1 increases the levels of dephosphorylated BimEL in MCF-7. (a through c) Western blot shows the expression level of the Bim isoforms (EL, extra long; L, long, and/or S, short) in cells treated with hormones plus and minus IGF-1 (a), with two apparent size variants of BimEL, a high-molecular-weight BimEL (top band, denoted by the dash) and a low-molecular-weight BimEL (bottom band, denoted by the arrow) (b, c). fold diff., fold difference in signal intensity of the lower-molecular-weight BimEL band divided by the signal intensity of the high-molecular-weight BimEL band by using the total MAPK signal intensity per lane as the loading control. (d, e) Western blot shows the accumulation of the upper Bim EL band when cells with active MEK1 are treated with the proteasome inhibitor MG132, and preferential loss of the upper BimEL band, concomitant with accumulation of the lower-molecular-weight Bim EL form after λ protein phosphatase treatment of the protein lysates. The λ protein phosphatase digestion (described in Materials and Methods) was carried out for 20 minutes (lanes 2 and 5) or 1 hour (lane 3) on protein lysates isolated from breast cancer cells undergoing E2 treatment for 24 hours. Immunoblotting determined the levels of BimEL and pMAPK1/2; total MAPK served as loading control. (f) Western blot shows that TAM- and/or U0126- treated cells show significantly higher levels of BimEL protein than do E2-treated cells, with the highest levels of dephosphorylated Bim EL, correlating directly to the cleavage of PARP detectable by 72 hours.

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