FIGURE 4.

CIH increases IRES activity. A, Western blot of pRG Di-cis constructs with (+) or without (−) CIH. NIH3T3 cells were transiently transfected with control pRG Di-cis (lanes I and II), pRG-R1 construct (lanes III and IV), pRG-R2 (lanes V and VI), and pRG-R3 constructs (lanes VII and VIII). CIH treatment of R1 and R3 constructs resulted in an appreciable increase in the expression of EGFP, indicative of increased IRES activity after CIH treatment. In control pRG-Di-cis construct (lanes I and II) and in R2 construct, no appreciable increase in EGFP expression was detectable after CIH treatment. Expression of RLuc of the respective constructs remained constant without (−) or with (+) CIH treatment. The β-actin protein band represents equal loading of protein samples. B, densitometric assessment of EGFP expression of three independent Western blots as shown in A. Data are expressed as mean ± S.E. (error bars) (*, p < 0.05). C, CIH treatment of cultured astrocytes shows a change in the expression pattern of HMBs over time with increasingly dominant expression of the HMB₀ isoform. D, quantitative analysis of Western blots of HMBs after CIH. Lower (HMB₀) was measured separately from the cumulative evaluation of both upper migrating forms (HMB₁ and HMB₂). ***, p < 0.001. n = 4 independent experiments.