JCS038430 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The effect of NAC or GSH on ER stress-induced induction of P450 2E1. Neo cells were treated with or without 1 mM NAC or GSH, and were then treated with 5 µM thapsigargin (upper) and 5 µg/ml tunicamycin (lower) for 6 hours. The cells were subsequently immunoblotted with anti-P450 2E1 and β-actin antibodies. THAP, thapsigargin; TUN, tunicamycin; NAC, N-acetyl-cysteine; GSH, reduced glutathione.

• Supplemental Figure S2 -

  Fig. S2. Effect of P450 2E1 on the accumulation of ROS and cell death in the BI-1 knockdown system. (A) HepG2 cells were transfected with non-specific and BI-1 siRNA. Sixteen hours later, the expression of P4502E1, BI-1 and β-actin were analyzed by western blot. (B) Non-specific and BI-1 siRNA-transfected HepG2 cells were treated with either 5 µM thapsigargin or 5 µg/ml tunicamycin for 20 hours, DCFDA was loaded into the cells and the fluorescence was measured. The amount of fluorescence of DCF in the presence of the ER stress agent was normalized compared with that of DCF without any stress agent. THAP, thapsigargin; TUN, tunicamycin; NS, non-specific siRNA. *,^#Significantly different from the fluorescence intensity of DCF in thapsigargin (*P<0.05) or tunicamycin (^#P<0.05)-treated Neo cells (Ns-siRNA-transfected). $,^&Significantly different from the fluorescence intensity of DCF in thapsigargin ($P<0.05) or tunicamycin (^&P<0.05)-treated BI-1 cells (Ns-siRNA-transfected).

• Supplemental Figure S3 -

  Fig. S3. Expression, purification and western blotting of BI-1. (A) 12% SDS-polyacrylamide gel electrophoresis (PAGE) of BI-1 fractions during purification. Lane 1: total extract of E. coli in the absence of 0.1 mM IPTG. Lane 2 and 3: induction of BI-1 by the addition of IPTG and purified BI-1 using a Hi-Trap Chelating column (Amersham Biosciences) charged with nickel ions according to the instructions of the manufacturer. Proteins were visualized by Coomassie brilliant blue staining. (B) Western blotting of purified BI-1 using primary antibodies against the C terminus of BI-1 (polyclonal antibody, 1:1,000 titer, lane 1) and the His₆ tag (monoclonal antibody, 1:500 titer, lane 2), respectively. Detection was performed by ECL. (C) 12% SDS-PAGE of purified BI-1 and its deletion mutants. GST represents 3 µg of glutathione-S-transferase as a control size marker. Δ8 and Δ16 represent BI-1 mutants deleted by 8 and 16 amino acids at the C terminus, respectively.

• Supplemental Figure S4 -

  Fig. S4. Emission spectra of Ac-Tempo. (A) The emission spectra of Ac-Tempo were recorded under the same procedure as that described for Amplex Red, with the addition of 5 µM Ac-Tempo into reaction samples. After further incubation at 37°C for 5 minutes, the fluorescence intensity was measured. (B) The λ_max (fluorescence intensity at 440 nm) was plotted with increasing BI-1 (or BI-1 deletion mutants)/NPR ratios. F.I. represents fluorescence intensity.

• Supplemental Table S1 -