Sc 390811

Tissue microarray (TMA) was constructed as described previously, and IHC analysis was performed as described previously 20 (link). Briefly, the prepared sections were incubated with GCLC antibody (1:100, sc-390811, Santa Cruz, CA, USA) at 4°C overnight. The horseradish peroxidase (HRP)-conjugated secondary antibody was incubated at 37°C for 45 minutes and then diaminobenzidine (DAB) solution (Dako REAL^tm EnVision TM Detection System, Denmark. Cat No#K5007) was applied. The nuclei were counterstained with Harris' Hematoxylin. PBS and monoclonal Mouse IgG (isotype control, 1:1000, Abacm, Cambridge, UK. Cat No#172730) were set as negative control. Two pathologists who were blinded to the clinical data independently assessed the IHC staining of GCLC using a semiquantitative histological score (H-score) approach, which combines the intensity and number of cells positive for GCLC expression. Staining intensity of GCLC was categorized as follows: 0 (-); 1 (+); 2(++) and 3(+++). The mean percentage of positively stained cells was scored as follows: 0 (<5%); 1 (5-25%); 2 (26-50%); 3 (51-75%) and 4 (76-100%). The final scores were generated by multiplying the staining proportion scores with staining intensity scores. Based on the final scores, GCLC expression levels were classified into four grade: negative (0), or weak (1-4), or moderate (6-8), or strong (9-12).

For immunoblotting, cells were washed with cold PBS for three times and lysed in the RIPA extraction regent (Pierce Biotechnology, Rockford, IL, USA) containing 1% protease inhibitor cocktail (Roche, France) and phosphatase inhibitor (Sigma, France) for 1 hour on ice. After clarified by centrifugation for 10min at 12,000×g, the supernatant of cell lysates was added with 5X SDS-PAGE loading buffer (Beyotime, Shanghai, China. Cat No#P0015) and then heated to 95-100°C for 10 minutes. 20µg total protein were loaded per well for blotting and electrotransfer to 0.45µm PVDF membrane (Immobion-P Transfer Membrane, Millipore, Billerica, MA, USA. Cat No# IPVH 00010). Following 5% non-fat dry milk blockage of membranes, specific primary antibody for GCLC (1:1000, sc-390811, Santa Cruz, CA, USA) and GAPDH (1:2000; Sigma-Aldrich, USA. Cat No# G9295) serving as internal reference were incubated overnight in 4°C. After washed three times for 5 minutes with TBST, the membranes were incubated with secondary antibody (Peroxidase-conjugated AffiniPure Goat anti-Mouse IgG (H+L), 1:2000; Jackson ImmunoResearch, USA. Cat No#111-035-003) for 2 hours and then exposed to Tanon-5200 Chemiluminescent Imaging System (Tanon, China) for imaging. All the experiments were performed for three times.