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. 2008 Mar 26;1:25–34. doi: 10.4137/bcbcr.s629

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© the author(s), publisher and licensee Libertas Academica Ltd.

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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Figure 3. — Protein expression of components of cell signaling pathways in MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Total protein was isolated 1, 3, and 5 days after transfection by RIPA. Western blotting was performed, and the membranes were probed with antibodies to the intermediates in the signaling pathways. (A) Western blots indicating β₂-adrenergic, CREB, phosphoCREB, Akt, PhosphoAkt, ERK, phosphoERK and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of signaling proteins being measured were standardized by dividing the densitometry of the control GAPDH (N = 3). Data were analyzed by ANOVA followed by the Tukey test. *indicates p < 0.05 from control. β₂-adrenergic protein expression was reduced from control levels at all three time periods. β₂-adrenergic phosphorylation was increased at 24 hours, but reduced at 3 days and 5 days by all three constructs (data not shown). CREB protein expression was reduced by two siRNA constructs at 24 hour (1104,1490) and 3 day (1104,1315), but only one construct at 5 days (1315). CREB phosphorylation was reduced by all constructs at 24 hours and 5 days, but increased by one construct at 3 days (1315). Akt protein expression was reduced by all constructs at 24 hours and 3 days, but only one construct at 5 days (1315). Akt phosphorylation was reduced at 24 hours and 3 days, but increased at 5 days for all constructs. ERK protein expression was reduced at 24 hours and 3 days by all constructs, but increased by one construct at 5 days (1104). ERK phosphorylation was increased at 24 hours (1104), but reduced by the other two constructs; reduced by two constructs at 3 days (1315, 1490); and increased by two constructs at 5 days (1315,1490). The bands are consistent with the expected size: β₂ (68 kDa); CREB/pCREB (43 kDa); Akt/pAkt (60 kDa); ERK/pERK (42–44 kDa); GAPDH (37 kDa). These are gels representative of two separate experiments.

Figure 3. — Protein expression of components of cell signaling pathways in MDA-MB-453 cells transfected with GIRK1 stealth siRNA. MDA-MB-453 breast cancer cells were transfected with 3 stealth siRNA constructs (Invitrogen). Total protein was isolated 1, 3, and 5 days after transfection by RIPA. Western blotting was performed, and the membranes were probed with antibodies to the intermediates in the signaling pathways. (A) Western blots indicating β₂-adrenergic, CREB, phosphoCREB, Akt, PhosphoAkt, ERK, phosphoERK and GAPDH expression of MDA-MB-453 cells transfected with three GIRK1 siRNA constructs (1104, 1315, 1490). Samples were collected at 24 hours, 3 days and 5 days. (B) Graphs indicate densitometry of the Western blots. The densitometry of signaling proteins being measured were standardized by dividing the densitometry of the control GAPDH (N = 3). Data were analyzed by ANOVA followed by the Tukey test. *indicates p < 0.05 from control. β₂-adrenergic protein expression was reduced from control levels at all three time periods. β₂-adrenergic phosphorylation was increased at 24 hours, but reduced at 3 days and 5 days by all three constructs (data not shown). CREB protein expression was reduced by two siRNA constructs at 24 hour (1104,1490) and 3 day (1104,1315), but only one construct at 5 days (1315). CREB phosphorylation was reduced by all constructs at 24 hours and 5 days, but increased by one construct at 3 days (1315). Akt protein expression was reduced by all constructs at 24 hours and 3 days, but only one construct at 5 days (1315). Akt phosphorylation was reduced at 24 hours and 3 days, but increased at 5 days for all constructs. ERK protein expression was reduced at 24 hours and 3 days by all constructs, but increased by one construct at 5 days (1104). ERK phosphorylation was increased at 24 hours (1104), but reduced by the other two constructs; reduced by two constructs at 3 days (1315, 1490); and increased by two constructs at 5 days (1315,1490). The bands are consistent with the expected size: β₂ (68 kDa); CREB/pCREB (43 kDa); Akt/pAkt (60 kDa); ERK/pERK (42–44 kDa); GAPDH (37 kDa). These are gels representative of two separate experiments.