Fig. 5.

The effect of PIM1 knockdown in LPS-mediated activation of TAK1 expression in macrophage-like THP-1 cells. (A) Whole cell lysates were isolated and used to measure the protein expression levels of TLR4 and MyD88 by Western blotting. (B) Cells were stimulated with LPS (1 µg/mL) for 6 h after pre-treatment with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h. Whole cell lysates were isolated and used to measure the protein expression levels of TLR4 and MyD88 by Western blotting. (C-E) Cells were transfected with control siRNA or PIM1 siRNA, and pre-treatment with TAK1 inhibitor (5Z)-7-Oxozeaenol (1 µM), or with PIM447 (20 µM) and AZD1208 (20 µM) for 1 h before LPS (1 µg/mL) stimulation. Whole cell lysates were isolated and used to measure the protein expression levels of p-TAK1, TAK1 and pro-IL-1β by Western blotting. Interaction of Pim-1 with TAK1 protein in LPS-induced THP-1 cells. (F) THP-1 cells were stimulated with LPS (1 µg/mL) for 6 h, and cell lysates were subjected to immunoprecipitation with TAK1, then the protein expression levels of Pim-1 and TAK1 were detected by Western blotting. (G) Cells were transfected with control siRNA or PIM1 siRNA, and Whole cell lysates were subjected to immunoprecipitation with TAK1, then the protein expression levels of Pim-1 and TAK1 were detected by Western blotting. (H) Cells were pre-treatment with 1 µM of TAK1 inhibitor (5Z)-7-Oxozeaenol for 1 h before LPS (1 µg/mL) stimulation. Whole cell lysates were subjected to immunoprecipitation with Pim-1, then the protein expression levels of TAK1 and Pim-1 were detected by Western blotting. (I, J) RAW 264.7 and BV2 cells were transiently transfected with plasmids expressing pMX-IRES-EGFP-Flag empty vector (control) or pMX-Pim-1 DN, pMX-Pim-1 WT, and pMX-Pim-1 K67M (mutant forms). The cell lysates were subjected to immunoprecipitation with anti-Flag, then the protein expression levels of TAK1 and Flag were detected by Western blotting