Fig. 2

IFN-γ mRNA and protein expression is not detectable during skin repair. (A) Total cellular RNA (20 µg) from normal and wounded back skin was analyzed by RNase protection assay for expression of IFN-γ. The complete absence of IFN-γ mRNA expression in normal Balb/c mice (upper panel, indicated as wt), or diabetic animals (lower panel, indicated as db) is shown. Sixteen wounds (n = 16) from the backs of four animals were excised for each experimental time point and used for RNA isolation. The time after injury is indicated at the top of each lane. Control skin refers to nonwounded skin of mice. One thousand cpm of the hybridization probe were added to the lane labeled probe. To verify the functionality of the used IFN-γ-antisense RNA probe, we hybridized the IFN-γ probe to total RNA isolated from the spleen of an endotoxin (LPS)-treated mouse (spleen, LPS). Expression of GAPDH mRNA is shown as a loading control for both experiments. (B) Total protein (50 µg) from lysates of non-wounded and wounded back skin (days 1, 3, 5, 7, and 13 after injury, indicated at the top of each lane) of Balb/c mice (indicated as wt), or db/db mice (indicated as db) were analyzed by immunoblotting for the presence of IFN-γ. Four wounds (n = 4) from the backs of four animals were excised for each experimental time point and used for protein isolation. We have used different amounts of recombinant murine IFN-γ as a positive control (indicated by an arrow). One representative immunoblot is shown for each condition.