Dc lowry assay

Frozen liver tissues (50–80mg) from mice were homogenized in lysis buffer (10ml/ g tissue) and protein concentration measured using the DC Lowry assay (Bio-Rad). Samples were prepared in Laemmli buffer and denatured at 97°C for 5 mins. Proteins were separated on 8 to 10% gels by SDS-PAGE and PVDF membranes were probed with antibodies against rabbit anti-Cyp2E1 (Abcam ab28146, 1:1000), rabbit anti-pEIF2α (Cell Signaling 3597, 1:1000) and rabbit anti-CHOP(Cell Signaling 5554, 1:1000). HSC-70 (mouse anti-HSC-70, Santa Cruz, sc-7298), was used as the loading control. Protein bands were visualized using ECL prime chemiluminescent development. Differences in chemiluminescent signal intensity were quantified using the ImageJ software.

Frozen cells were resuspended in room-temperature 25 mM PIPES, 250 mM NaCl, pH 7.2 and lysed by sonication (1 s on, 1 s off, 10 min, 90% power setting). The cell debris was removed, and the membranes were isolated and washed as described previously.^{12 (link)} The washed membranes were then resuspended in the PIPES buffer to 300 μM total protein, as determined by the Bio-Rad DC Lowry assay (with BSA as a standard), aliquoted in X-band and Q-band EPR tubes, and frozen in liquid nitrogen until analysis. Membranes were aliquoted, flash frozen in liquid nitrogen, and stored at −80 °C until further purification. Mc. sp. str. Rockwell pMMO was further purified and solubilized as described previously, but with PIPES instead of Tris buffer to avoid copper chelation by the buffer.^{12 (link)} In brief, membranes were solubilized with n-dodecyl β-d-maltopyranoside (DDM), diluted with 25 mM PIPES, 0 M NaCl buffer, pH 7.2, and centrifuged at 100 000g for 1 h, and the soluble fraction was loaded onto a Source 15Q column (GE Healthcare). Fractions were assessed for purity by SDS PAGE, and those deemed pure were collected, concentrated, and flash frozen for later use. The protein concentration was determined by measuring the absorbance at 280 nm and using the extinction coefficient 7.66 × 10³ M⁻¹ cm⁻¹.^{12 (link)}