Fig. 6

Co-immunoprecipitation and immunoblot analyses of RP105 and its signaling partners on bone marrow monocytes in response to LPS in Ceacam1^−/− and wild type mice (a) Immunoblot analysis of RP105, MD1 and Syk after immunoprecipitation (IP) with RP105 antibody in WT and Ceacam1^−/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min. (b) Immunoblot analysis of RP105, pVAV1 and β-actin after IP with RP105 antibody in WT and Ceacam1^−/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). (c) Immunoblot analysis of CEACAM1 and β-actin after IP with CEACAM1 antibody in WT bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). (d) IL-6 mRNA expression of bone marrow monocytes pretreated with pVAV1 inhibitor azathioprine and 6-thio-GTP for 20 min following 500 ng/mL LPS treatment for 30 min in vitro (n = 3). (e) IL-6 mRNA expression of bone marrow monocytes treated with RP105 activating antibody (clone RP/14, 20 μg/mL) or 500 ng/mL LPS for 30 min (n = 3). (f) IL-6 mRNA expression of bone marrow monocytes treated with blocking CD14 antibody (clone M14-23, 10 μg/mL) or blocking MD1 antibody (clone MD113, 10 μg/mL) for 20 min following 500 ng/mL LPS for 30 min (n = 3). (g) Immunoblot analysis of CD14 and Src after IP with RP105 antibody in WT and Ceacam1^−/− bone marrow monocytes with or without 500 ng/mL LPS treatment for 15 min (TL: total cell lysate). (h) IL-6 mRNA expression of bone marrow monocytes pretreated with 50 nM Src l1 (Src inhibitor) for 20 min following treatment of 500 ng/mL LPS for 30 min (n = 3). (i) Anti-CEACAM1 IPs from bone marrow monocytes after treatment with 500 ng/mL LPS for 15 min were followed by immunoblot analysis with 4G10 or anti-pSHP1 antibodies. (Upper); IP with anti-CEACAM1, immunoblot analysis with 4G10. Loading control: immunoblot with anti-CEACAM1. (Lower): IP with anti-CEACAM1 and immunoblot with anti-pSHP-1. Loading control: immunoblot with anti-SHP1. (TL: total cell lysate)