Complete and phostop

Total protein from liver tissues and cell co-cultures were processed in the ice-cold lysis buffer containing 20 mM Tris-Cl, pH 7.8, 137 mM NaCl, 1% (v/v) Nonidet P40, 10% (v/v) glycerol, 2 mM EDTA (All from Sigma; St. Louis, MO), supplied with Complete and PhoSTOP (Roche, Branchburg, NJ) as previously described [14 (link)]. Proteins of interest were immunoprecitated with protein A/G Plus-Agarose complex beads (sc2003, Santa Cruz Biotechnology, Santa Cruz, CA) and either mouse anti-Dvl or mouse anti-DVL3 for mice and co-culture samples, respectively. Co-precipitated were normalized by reprobing the blots to detect the constant regions (heavy chains) of the immunoglobulins used for IP assay [26 (link)].

Proteins extracted either from liver tissues or cell co-cultures were solubilized with the ice-cold lysis buffer containing 20 mM Tris-Cl, pH 7.8, 137 mM NaCl, 1% (v/v) Nonidet P40, 10% (v/v) glycerol, 2 mM EDTA (All from Sigma; St. Louis, MO), supplied with Complete and PhoSTOP (Roche, Branchburg, NJ) as previously described [23 (link)]. FLII and MYD88 proteins were immunoprecitated with protein A/G Plus-Agarose beads (sc2003, Santa Cruz Biotechnol.) in complex with either anti-FLII (rabbit-H300, sc30046) or anti-MYD88 (mouse-E11, sc74532) antibodies, respectively. After IP assays, FLII and MYD88 protein were immunodetected by western blot analyses with anti-FLII (muse-116.40, sc21716) and anti-MYD88 (goat-N19, sc8196) antibodies, respectively; while NXN and TLR4 were immunodetected using anti-NXN (GTX107039) and anti-TLR4 (25, sc-293072). Co-precipitated were normalized by reprobing the blots to detect the constant regions (heavy chains) of the immunoglobulins used for IP assays, as previously indicated [24 (link)].