Figure 9.

In vitro synthesis of a truncated HLA class I heavy chain by HLA-A2 mRNA with exon 2 skipping isolated from 624MEL28 cells. The cloned HLA-A2 cDNA lacking exon 2 sequence (E2⁻ A2) was used as a template for mRNA synthesis. The synthesized mRNA was translated in the presence of [³⁵S]methionine in a coupled transcription/translation system driven by T₇ promoter (Promega Corp.). A wild-type HLA-A2 cDNA (A2) was used as a control. Translation products were analyzed by SDS-PAGE (A) or were immunoprecipitated by the rabbit anti–β₂-μ–free HLA class I heavy chain serum R5996-4 and by the anti–β₂-μ–free HLA heavy chain mAb HC-A2 before analysis by SDS-PAGE (B). Normal rabbit serum (NRS) was used as a negative control. The degree of translation was quantitated by scanning densitometry of the intrinsically radiolabeled proteins in the gel (C). The level of RNA synthesis was measured by [³²P]UTP incorporation obtained from TCA precipitation (D).