Anti c src

Western blotting was performed as previous reported [36 (link), 37 (link)]. Briefly, Hep-2 cells were grown in the mid-log phase, washed with phosphate buffered saline and lysed for 30 min on ice. The lysis buffer with pH 6.8 contained 10% glycerol, 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mmol/L Tris-HCl. The SDS–PAGE analysis was performed and membranes were incubated overnight at 4°C with antibodies directed against the indicated proteins. The antibodies were obtained from the following companies: anti-TrkB, anti-β-actin, anti-p-Akt, anti-cyclin D1, anti-p-s-Src and anti-c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-RUNX3 and anti-Keap1 were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Twist-1 and anti-Twist-2 were purchased from BD Biosciences (San Jose, CA, USA). Membranes were washed, incubated with the appropriate secondary antibodies and exposed with the ECL chemiluminescent substrate kit (Pierce, Rockford, IL, USA). Images were analyzed with ImagePro (Media Cybernetics, Bethesda, MD, USA) and densitometry data were analyzed by using either conventional Student’s t-test. Results are reported as mean ± SD. A P-value <0.05 was considered significant and all were two-tailed.

To analyze activity of upstream kinases, c-Abl or c-Src was immunoprecipitated from 500 μg of whole-cell lysate using 2 μg of anti-c-Src (Santa Cruz, Germany) or 2 μg of anti-c-Abl antibody (24-11; Santa Cruz, Germany). Beads were washed twice in lysis buffer and twice in kinase assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl₂, 10 mM MnCl₂, and 2 mM dithiothreitol [DTT]). Immunoprecipitated c-Abl was analyzed separately by collecting one-fifth of the bead slurry directly after the washing steps. For in vitro kinase reactions of c-Src, H. pylori lysate containing CagA was used as a substrate. Briefly, H. pylori was sonicated in kinase buffer and centrifuged at 20,000 × g. Ten micrograms of cleared H. pylori WT or H. pylori ΔcagA lysate was added as a kinase substrate. For in vitro kinase reactions of c-Abl, 1 μg of recombinant glutathione S-transferase (GST)-CrkII WT (aa 120 to 225) (32 (link)) was added to the washed antigen-coupled beads together with 250 μM ATP and incubated for 30 min at 30°C and 1,000 rpm on a thermomixer (Eppendorf, Germany). Recombinant GST-CrkII (aa 120 to 212) was included as a negative control (32 (link)). To stop the reaction, 4× sample buffer was directly added to the samples and immediately boiled at 95°C for 7 min and subjected to Western blot analysis.