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. 2009 Sep 2;284(44):30615–30626. doi: 10.1074/jbc.M109.041244

FIGURE 4.

FIGURE 4.

LPS stimulates the proteolytic pathway in the macrophages. A, LPS up-regulates BG21 in the macrophages. Left, BMDM and RAW264.7 macrophage cells were left intact or treated 24 h with LPS. Total RNA extracted from the cells was analyzed by Q-RT-PCR to determine the levels of the BG21 and J37 mRNAs. Right, murine breast carcinoma 4T1 and macrophage RAW264.7 cells were treated with LPS for the indicated time. The cell lysate samples (50 μg of total protein) were analyzed by Western blotting with the Golli-MBP antibody. The three right lanes show the in vitro cleavage of BG21 by MMP-25 at a 1:100–1:1000 enzyme/substrate molar ratio and intact BG21. The experiments were repeated 3–5 times, and the data represent the mean ± S.E. *, p < 0.05. B, LPS up-regulates MMP-25 in the macrophages. Left, RAW264.7 cells were left intact (−LPS) or treated 24 h with LPS (+LPS). Total RNA extracted from the cells was analyzed by RT-PCR to determine the levels of the mRNAs of the individual MMPs. GAPDH was used as a control. The calculated size of the amplified fragments was 278, 343, 297, 285, 299, and 300 bp for MMP-2, -9, -12, -14, -25, and GAPDH, respectively. Right, two independent RAW264.7 cell samples were left intact or treated 24 h with LPS. The cell lysate samples (50 μg of total protein) were analyzed by Western blotting with the MMP-25 antibody (top panel). The images were scanned and digitized using the Fujifilm MultiGauge software. The MMP-25 band density (bottom panel) is shown in arbitrary units (AU). These experiments were repeated 3–5 times with comparable results. *, p < 0.05. C, LPS up-regulates PC2 in the macrophages. BMDM and RAW264.7 cells were left intact or treated with LPS for 18–36 h. The cell lysate samples (50 μg of total protein) were analyzed by Western blotting with the PC2 antibody (top panel). Murine mammary carcinoma 4T1 cells (4T1), which do not produce any significant level of PC2, were used as a negative control. The images were scanned and digitized using the Fujifilm MultiGauge software. The PC2 band density (bottom panel) is shown in arbitrary units (AU). These experiments were repeated 3–5 times with comparable results. *, p < 0.05.