Western blot chemiluminescence reagent

Tissue homogenates were lysed in the lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 2 mM Na₃VO₄, 10 mM NaF, 10 mM β-glycerophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, and 2 mM phenylmethylsulfonyl fluoride). Cultured cells were harvested and lysed in the lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2 mM Na₃VO₄, 10 mM NaF, 10 mM β-glycerophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, and 2 mM phenylmethylsulfonyl fluoride). Lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and subjected to immunoblotting. Proteins were visualized using western blot chemiluminescence reagent (PerkinElmer Life Sciences). Intensity of chemiluminescence was quantified using the LAS4000 system (FUJIFILM).

Cells were harvested and lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 mM Na₃VO₄, 10 mM NaF, 10 mM β-glycerophosphate, 2 mM PMSF, 10 μg/mL leupeptin, 10 μg/mL trypsin inhibitor, 10 μg/mL aprotinin, 1% NP-40). Samples were run on SDS-PAGE gel, and separated proteins were transferred to PVDF membranes (Merck Millipore, Country Cork, Ireland). After transfer, membranes were treated with a blocking solution (1% BSA or 5% non-fat milk in 0.1% Tween 20-TBS). The membranes were incubated with primary antibodies. Anti-β-actin antibody (8H10D10), anti-hemagglutinin (HA) antibody (6E2), anti-CEACAM1 anrtibody (D3R80), and anti-CECAM1 (D1P4T) antibody were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-phosphotyrosine antibody (4G10) was obtained from Millipore (Darmstadt, Germany); anti-HA antibody (3F10) was obtained from Roche (Mannheim, Germany), and anti-CagA antibody (HPP-5003-9) was obtained from Austral Biologicals (San Ramon, CA, USA). Secondary antibodies used were conjugated with horseradish peroxidase (HRP). Protein bands were visualized by Western blot chemiluminescence reagent (Perkin Elmer Life Sciences, Waltham, MA, USA).

Cells were collected and lysed in lysis buffer containing 250 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.5% NP-40, 2 mM Na₃VO₄, 10 mg ml⁻¹ leupeptin, 10 mg ml⁻¹ trypsin inhibitor, 10 mg ml⁻¹ aprotinin and 2 mM phenylmethylsulfonyl fluoride. For immunoprecipitation, cell lysates were incubated with respective antibodies and protein G-beads (GE Healthcare). The beads were then washed six times with the lysis buffer, and the immune complex was eluted with SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer. The lysates and immunoprecipitates were subjected to SDS–PAGE followed by immunoblotting. Proteins were transferred to a polyvinylidene difluoride membrane filter (Millipore) and visualized using western blot chemiluminescence reagent (Perkin-Elmer Life Sciences). The obtained chemiluminescence was exposed to X-ray film (GE Healthcare). Uncropped images for immunoblots are shown in Supplementary Fig. 8.