Any kd tgx gels

To analyze Lm‐γ2 fragments produced by cultured human cancer cell lines, conditioned medium was collected from confluent culture of each cell line after 2 days of incubation in serum‐free medium, and treated with 10% (w/v) trichloroacetic acid to precipitate protein. The protein obtained from 1 mL conditioned medium was applied to SDS‐PAGE. To analyze γ2 fragments produced in cancer tissues, two serial frozen sections with an approximate size of 5 × 5 × 0.005 mm were combined and extracted with 100 μL lysis buffer containing 20 mM Tris‐HCl (pH 7.5), 1% (v/v) Triton X‐100, 1 mM EDTA, and protease inhibitors and centrifuged at 21 500 × g for 10 min. Protein in the resultant supernatant was precipitated with 4 volumes of cold acetone and dissolved in 50 μL SDS sample buffer (soluble fraction), while the precipitate was again extracted with 50 μL SDS sample buffer (insoluble fraction). Twenty microliters of each fraction was applied to SDS‐PAGE. The electrophoresis was carried out on Any kD TGX gels (Bio‐Rad, Hercules, CA, USA) under reducing conditions. For immunoblotting, proteins separated by SDS‐PAGE were transferred onto PVDF membranes, probed with indicated antibodies, and visualized by the ECL method (GE Healthcare, Little Chalfont, UK).