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. 2010 Jun 14;30(16):3929–3942. doi: 10.1128/MCB.00087-10

FIG. 3.

FIG. 3.

TLR4 signaling is not affected in Gfi1−/− BMDMs. Wild-type (WT) and Gfi1−/− BMDMs were treated with medium or 10 ng/ml of LPS, and cells were harvested at the indicated time points. (A) Flow cytometry of TLR4-MD2 expression of medium-treated (−LPS) and LPS-stimulated (+LPS) wild-type (WT) and Gfi1−/− BMDMs. Staining with irrelevant antibody was used as a control. (B) Expression levels of IRAK-M and SHIP1 determined using RT-PCR. (C) The activation levels of p-Akt, p-Erk, p-JNK, p-p38, and p-IκBα were assessed using immunoblotting. Immunoblot analysis of total endogenous proteins of each signaling molecule was used to ensure equal sample loading. (D) Extracts from WT and Gfi1−/− BMDMs treated as indicated were probed with antibodies against NF-κB subunit p65 or the phosphorylated form of p65 (p-p65). (E) Nuclear extracts of wild-type and Gfi1−/− BMDMs treated as indicated were probed with antibodies against the NF-κB subunit p65. (F) WT and (G) Gfi1−/− BMDMs were treated with 10 ng/ml LPS for the indicated times, and the localization of endogenous NF-κB p65 was assessed by confocal microscopy. Nuclei were visualized by Topro3 staining.