Western Blot Analysis of Abl Signaling

Cells were lysed in 20 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 x complete protease inhibitor tablets (Roche Diagnostics, Germany), 1 mM sodium molybdate, 20 mM NaF, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 1 mM sodium vanadate. Equal protein amounts were separated by SDS PAGE and transferred onto nitrocellulose. Following antibodies were used: anti-c-Abl (AB3, Merck Biosciences, Germany), anti-pAbl^T735, anti-pCrkII^Y221 (both New England Biolabs, Germany), anti-pAbl^Y245, anti-β-actin (both Sigma Aldrich, Germany), anti-pAbl^Y412, anti-GAPDH (both Abcam, UK), anti-CagA [44 (link)], anti-GST (Biomol Germany), anti-14-3-3 H8, anti-phospho-tyrosine (pY99), anti-TTK, and anti-PKC (all Santa Cruz Biotechnology, Germany). Membranes were imaged using the Molecular Imager ChemiDoc XRS system (BioRad, Germany). Where indicated, signals of protein bands were quantified using the ImageLab software (BioRad, Germany).

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Posselt G., Wiesauer M., Chichirau B.E., Engler D., Krisch L.M., Gadermaier G., Briza P., Schneider S., Boccellato F., Meyer T.F., Hauser-Kronberger C., Neureiter D., Müller A, & Wessler S. (2019). Helicobacter pylori-controlled c-Abl localization promotes cell migration and limits apoptosis. Cell Communication and Signaling : CCS, 17, 10.

Publication 2019

complete protease inhibitor tablets anti-β-actin anti-GAPDH anti-PKC Molecular Imager ChemiDoc XRS system ImageLab software

14 3 3 Actin Antibodies Cells Diagnostics Edta Gapdh Membranes Nacl Nitrocellulose Protease inhibitor Protein Sds page Sodium molybdate Sodium pyrophosphate Sodium vanadate Tris Triton x 100 Tyrosine β glycerophosphate

Corresponding Organization :

Other organizations : University of Salzburg, University of Zurich, Paul Ehrlich Institut, Max Planck Institute for Infection Biology, Charité - Universitätsmedizin Berlin, Paracelsus Medical University

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Variable analysis

independent variables

Lysis buffer components: 20 mM Tris pH 7.5, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 x complete protease inhibitor tablets (Roche Diagnostics, Germany), 1 mM sodium molybdate, 20 mM NaF, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 1 mM sodium vanadate

dependent variables

Protein expression and phosphorylation levels of: c-Abl, pAbl^T735, pCrkII^Y221, pAbl^Y245, pAbl^Y412, CagA, GST, 14-3-3 H8, phospho-tyrosine (pY99), TTK, and PKC

control variables

Equal protein amounts were separated by SDS PAGE and transferred onto nitrocellulose
Anti-β-actin and anti-GAPDH antibodies were used as loading controls

controls

Positive controls: Not specified
Negative controls: Not specified

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