Figure 1.

Effects of pemphigus and normal human IgG fractions on apoptosis-related molecules in keratinocytes. At ∼80% of confluence, keratinocyte monolayers from three foreskin donors (n = 3) were exposed to 1) 1 mg/ml normal pooled human sera, NIgG; 2) IgG fractions isolated from the serum of PV-1 or PV-2 patient with active disease; 3) sera of these PV patients after a course of IVIg therapy showing improvement; 4) the IVIg batches used for transfusion of each patient, IVIgG; or 5) preincubated for 1 hour with an IVIgG fraction and then exposed to corresponding PVIgG-1b or PVIgG-2b. After incubation at 5% CO₂, the effects of experimental treatments on the relative amounts of mRNA and proteins of the pro- and anti-apoptotic molecules were quantified by real-time PCR and Western blot, respectively. Asterisks indicate significant (P < 0.05) differences from control. A: Real-time PCR analysis. The real-time PCR was performed using RNA isolated from keratinocytes incubated with test IgGs for either 12 hours (dashed bars) or 24 hours (open bars) exactly as described in Materials and Methods. The alterations in the gene expression levels of caspases 3 and 8, Fas-L, Fas-R, and FLIP-l are presented relative to the rates of expression of corresponding genes in control samples, taken as the baseline. B: Western blot analysis. After 24 hours of exposure, the protein levels of caspases 3 and 8, Fas-L, Fas-R, and FLIP-l were analyzed by Western blot. The gene expression ratio of 1 was assigned to control, non-treated monolayers (C). The images show typical bands appearing at the expected molecular weight (MW) indicated to the right of the gels. The ratio data underneath the bands are the means ± SD of the values obtained in three independent experiments. Specific staining was absent in the negative control experiments in which the membranes were treated without primary antibody or with irrelevant primary antibody of the same isotype and host (not shown).