Protein a g

Cell lysates were made in lysis buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol) and a mixture of proteases inhibitors (Protease Inhibitor Cocktail, Roche) according to the manufacturer’s instructions. Proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences Corp., Piscataway, NJ, USA). Blots were developed using the enhanced chemiluminescen (ECL) system (Amersham Biosciences Corp.). For co-immunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Amersham Biosciences) for 2 h at 4 °C. Immunocomplexes were extensively washed, resolved by SDS–PAGE, and analyzed by immunoblot assay. Antisera and monoclonal antibodies were the following: anti-FLAG, anti-β-Actin, (Sigma-Aldrich, St. Louis, MI, USA); anti-HA, anti-myc, anti-MALT1, anti-CARMA2, anti-NEMO, anti-ubiquitin, (Santa Cruz Biotechnology, Dallas, TX, USA); and anti-RNF7 (Abcam, Cambridge, UK). The anti-BCL10 antibody was described in [37 (link)].

Cell lysates were made in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 1% Triton X-100, 10% glycerol) and a mixture of proteases inhibitors (Protease Inhibitor Cocktail, Roche) according to the manufacturer’s instructions. Proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Blots were developed using the ECL system (Amersham Biosciences). For co-immunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Amersham Biosciences) for 2 hrs at 4°C. immunocomplexes were extensively washed, resolved by SDS–PAGE, and analyzed by immunoblot assay. Sources of antisera and monoclonal antibodies were the following: anti-FLAG, anti-β-Actin, Sigma; anti-HA and anti-BCL10 (H-197 SC5611, generated against an epitope corresponding to amino acids 1–197 of human BCL10), Santa Cruz Biotechnology. The calf-intestinal alkaline phosphatase was purchased from Roche.