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. 2011 Aug 18;286(41):35407–35417. doi: 10.1074/jbc.M110.205708

FIGURE 4.

FIGURE 4.

Knockdown of GPx-1 and NFκB and MAPK signaling pathways in TNF-α-treated cells. A, to determine the effect of TNF-α on NFκB signaling in GPx-1-deficient and control cells by immunoblotting, nuclear extraction was used to detect NFκB p50 and USF-2 protein (a nuclear control) and the cytoplasmic fraction for IκBα and actin protein (cytoplasmic control) (n = 3). B, qRT-PCR was used to measure NFκB1 mRNA after 10 ng/ml of TNF-α for 15 min and 2 h in GPx-1-deficient and control cells. UT indicates untransfected cells. mRNA was measured by qRT-PCR following normalization to GAPDH as endogenous control and compared with an untransfected sample to obtain relative mRNA levels. Significant differences were observed between transfected cells analyzed by pairwise comparison with the Fisher's PLSD test (*, p < 0.05, n = 4). C and D, Western blot analysis: C, the effect of TNF-α on MAPK signaling was tested by analyzing phospho-ERK1/2 protein and phospho-JNK in GPx-1-deficient and control cells (n = 4). D, GPx-1-deficient and control cells were pretreated with MEK1/2 inhibitor (U0126) 1 h prior to TNF-α exposure at 10 ng/ml for 4 h. E, GPx-1-deficient and control cells were pretreated with a JNK inhibitor (SP600125) 1 h prior to TNF-α exposure. F, GPx-1-deficient and control cells were pretreated with PDTC, an NFκB inhibitor, immediately following transfection. Shown is the effect of PDTC on ICAM-1 and VCAM-1 in the absence or presence of TNF-α (1 ng or 10 ng/ml).

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