Immunoprecipitation of CRT, c-Myc, and Integrins

For immunoprecipitation, as described previously ^{58 (link)}, whole protein lysates (prepared from Capan-2 and AsPC-1 cells) were extracted in a lysis buffer (20 mM Tris/HCl, pH7.4, 1.0% NP-40, 1 mM EDTA, 150 mM NaCl, 50 μg/ml PMSF, 10 μg/ml leupeptin). Briefly, CRT, c-Myc and IgG (Santa Cruz) antibodies were preincubated with magnetic beads (Bio-Rad, Hercules, CA, USA) for 4 h at 4 °C. The antibody–beads complex was washed three times with the lysis buffer and incubated with the soluble supernatants of protein lysates overnight at 4 °C. Next, the immunocomplexes were washed three times with lysis buffer, eluted by boiling in sample loading buffer for SDS-PAGE and then subjected to WB analysis with CRT, c-Myc, Integrinβ1, Fibronectin and GAPDH antibodies. Each experiment was repeated three times.