Figure 4
The combination of CNTF and soluble CNTFRα increases Cox-2 and PGE 2 production in murine microglia that is not inhibited by a gp130 function blocking antibody. After cytokine treatment, cultured murine microglia were collected and 10 micrograms of protein lysates were analyzed by western blotting. A, Murine microglia cultures were treated with CNTF (10 ng/mL) (CN), the combination of CNTF (10 ng/mL) and soluble CNTFRα (200 ng/mL) (CN+sR), soluble CNTFRα (200 ng/mL) (sR), or left untouched (UT) for 16–18 hours. Membranes were probed with Cox-2 antibody and reprobed with β-tubulin antibody to confirm equivalent protein loading. Data are representative of 3 independent experiments. B, Murine microglia were treated with the combination of CNTF (5 ng/mL) and soluble CNTFRα (200 ng/mL) (CN+sR) or left untouched (UT) for 16–18 h. Supernatants were collected and analyzed by PGE2 ELISA. Values represent the means ± S.E.M. from 4 independent experiments. *p < 0.05 by Student's t-test. C, Murine microglia were treated with CNTF 0.4, 2, 10, 25 and 50 ng/mL in combination with soluble CNTFRα (200 ng/mL), or with LPS 0.1 ng/mL for 16–18 hours. Membranes were probed with Cox-2 antibody and reprobed with β-tubulin antibody. Data are representative of two independent experiments. D, Microglia were treated with gp130 antibody (5 μg/mL) (ab), the combination of CNTF (10 ng/mL) and soluble CNTFRα (200 ng/mL) (CN+sR), gp130 antibody for 1 hour followed by the combination of CNTF (10 ng/mL) and soluble CNTFRα (200 ng/mL) (ab+CN+sR), or left untreated (UT) for 18 h. Membranes were probed with Cox-2 antibody and reprobed with β-tubulin antibody. Data are representative of 4 independent experiments. E, Microglia were treated with gp130 antibody (5 μg/mL) (ab), IL-6 (5 ng/mL), a combination of IL-6 (5 ng/mL) and soluble IL-6R (200 ng/mL) (IL-6+sR), gp130 antibody for 1 hour followed by IL-6 (5 ng/mL) (ab+IL-6) or a combination of IL-6 (5 ng/mL) and soluble IL-6R (200 ng/mL) (ab+IL-6+sR), or left untreated (UT) for 20 minutes. Membranes were probed with phospho-STAT3 tyr705 antibody, stripped, and re-probed with STAT3 antibody.