Figure 2. Generation of the RNF146 reporter construct.

(A) Schematic diagram of the luciferase construct with the 1.9 kb human RNF146 promoter (pGL3-RNF146-Luc). The experimental schedule for H₂O₂ preconditioning is illustrated in the bottom panel. SH-SY5Y cells were treated with 100 uM H₂O₂ for 10 min, washed briefly, and maintained in complete medium. Luciferase assay, total RNA isolation, or protein extract preparation was performed at the indicated time points. (B) Relative RNF146 promoter activities in SH-SY5Y cells at the indicated time points following preconditioning (100 uM H₂O₂,10 min), as determined by the luciferase assay (n = 3). SH-SY5Y cells were cotransfected with pGL3-RNF146-Luc and pRL-TK for 24 hrs, followed by low-dose H₂O₂ preconditioning. (C) Quantification by RT-qPCR of relative RNF146 messenger RNA levels (normalized to GAPDH) in SH-SY5Y cells at the indicated time points after low-dose H₂O₂ preconditioning (100 uM H₂O₂,10 min, n = 3). (D) Representative western blot showing RNF146 expression in SH-SY5Y cells at the indicated time points following H₂O₂ preconditioning (100 uM H₂O₂,10 min). β-actin serves as a loading control. Quantification of relative RNF146 protein levels normalized to those of β-actin at the indicated time points after H₂O₂ preconditioning (n = 3). Data are expressed as mean ± SEM. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, ANOVA test followed by Tukey post hoc analysis.