Sc 55561

After boiling in Laemmli′s sample buffer, proteins (40 μg) were resolved on 7%, 12% or 15% SDS-polyacrylamide gels, and transferred to the PVDF membrane. Membranes were blocked with 5% BSA, and incubated with primary antibody. CTR1, DMT1, ATOX1, COX17, COX2 and CCS were detected using Santa Cruz Biotechnology (Santa Cruz, CA, USA) antibodies: sc-18473, sc-166884, sc-398742, sc-393617, sc-514489, sc-55561, respectively. SOD1, SOD2, CAT, GR and GPX, were detected using Abcam (Trumpington, Cambridge, UK) antibodies: ab13498, ab13533, ab16731, ab16801, ab22604, respectively. Anti-MT-1/MT-2 antibody (M0639) was the product of Dako (Agilent, Santa Clara, CA, USA). β-actin was detected by AC-15 antibody (Sigma-Aldrich, St. Louis, MO, USA). The blots were incubated with anti-rabbit, anti-goat, or anti-mouse secondary antibodies conjugated with horseradish peroxidase. Immunoreactive proteins were visualized by the enhanced chemifluorescence method. Quantitative analysis of immunoreactive bands was done using iBright^TM FL1500 Imaging System Software (Thermo Fisher Scientific, Waltham, MA, USA). All experimental samples and controls used for one comparative analysis are run on the same blot/gel.

After stimulations, HepG2 cells were washed with DPBS and lysed with 60 μl RIPA buffer. Whole cell lysates were centrifuged at 15,000 rpm at 4°C for 1 h. The supernatant was collected and stored at −20°C. Protein quantification was determined using the BCA assay. 20 μg of protein sample was loaded into 15-well SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated at 4°C overnight with primary antibodies. Primary antibodies used were anti-CCS (1:2000, sc-55561, Santa Cruz Biotechnology, anti-mouse), and anti-α-tubulin (1:10,000, MA1-80017, ThermoFisher, anti-rat). Primary antibodies were removed, and the membranes were washed with tris buffered saline with Tween (TBST). Membranes were then incubated in secondary antibodies at room temperature for 1 h. Secondary antibodies used were anti-mouse IgG AlexaFluor800 (A32789, 1:5000 Invitrogen) and anti-rat IgG (A21428, 1:5000 Invitrogen), AlexaFluor647, and a horseradish peroxidase-conjugated secondary antibody. Blot images were recorded on a BioRad ChemiDoc imaging system and processed in ImageLab.