FIGURE 1.

HER-2/neu-mediated reduced MHC class I surface molecules associated with low tapasin expression. A, flow cytometric analysis of H-2L^q surface antigen expression. 1 × 10⁵ cells were subjected to flow cytometry as described under “Experimental Procedures” using an H-2L^q and H-2L^d cross-reactive mAb and a respective murine isotype control. The upper histograms show the staining with isotype control antibody, and the lower histograms display the mean specific fluorescence intensity (MFI) of H-2L^q on the cell surface of HER-2/neu⁻ cells (left) and HER-2/neu⁺ cells (right). B, analysis of the tapasin protein expression in HER-2/neu⁻ and HER-2/neu⁺ cells. 30 μg of protein extract/lane was separated by SDS-PAGE under denaturating and reducing conditions, and protein expression was assessed by Western blot analysis as described under “Experimental Procedures” using an anti-tapasin-specific antibody. Staining of the same blot with GAPDH as a housekeeping gene served as control. Error bars indicate S.D. C, reduced APM transcription in HER-2/neu⁺ cells. Quantitative RT-PCR analyses of tapasin, TAP1, and TAP2 were performed with oligo(dT)-primed cDNA from HER-2/neu⁻ cells (black bars) and of HER-2/neu⁺ cells (white bars) and normalized to EF-1α transcription. Transcriptional levels from HER-2/neu⁻ cells were used as control and set to 1, whereas the transcripts of HER-2/neu⁺ cells (white bars) were directly compared. Data shown represent three independently analyzed biological samples.