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Figure S1. TPSNΔEx3 (primers 5 and 6 in Table 1) and tapasin (primers 7 and 8 in Table 1) specific primers for absolute quantification in qRT-PCR bind specifically. A. Dilution series of fitting templates for tapasin and TPSNΔEx3 including a nonfitting template (TPSNΔEx3 for tapasin and tapasin for TPSNΔEx3) shown as a solid gray line. Dilution series (number of linearized cDNA templates) were performed in 500 ng RNA extracted from .220 cells. Primer binding sites are schematically depicted.

Figure S2. Tapasin- and TPSNΔEx3 -specific RT-PCRs (primer 5 and 6 for tapasin, and 7 and 8 for TPSNΔEx3, Table 3) were performed using RNA from MRC5 cells stably transduced with tapasin-minigene constructs (WT and MUT). Diagram shows fold of transcripts compared to control transduced cells.

Figure S3. TPSNΔEx3 is not able to restore cell surface expression of HLA-B*44:02 in the absence of tapasin. (A) .220-HLA-B*44:02 cells tranduced with tapasin-, TPSNΔEx3- or ctrl-IRES-EGFP expressing lentiviral vectors were stained with TT4-A20 or W6/32. By FACS, EGFP positive cells were analyzed for HLA-B*44:02 cell surface expression. (B) .220-HLA-B*44:02 cells were electroporated with tapasin- or TPSNΔEx3 (NeTT)-expressing pcDNA3.1 and analyzed by fluorescence microscopy using TPN-C antibodies for staining of tapasin and TPSNΔEx3 and the mAb HC10 for MHC I.

Figure S4. (A) Long exposure of experiment shown in Fig. 4B. (B) Performed as in A using anti-ERp57, anti-calreticulin or control IgG antibodies (mouse anti-HA, Sigma) for the first IP followed by a re-IP with anti-TPN-C antibodies. In addition to analysis of the re-IP samples, the remaining PAS after dissociation of the 1st IP was applied to an SDS-PAGE.

Figure S5. HeLa cells were transiently co-transfected with tapasin-, TPSNΔEx3- or a control-IRES-EGFP expression plasmid together with HLA-B*44:02 or HLA-B*44:05 in pcDNA3.1. The transfection efficiency was ca 20%. At 18 hrs post-transfection cells were stained with the mAb TT4-A20 and anti-CD29. Histograms show TT4-A20 and CD29 stains of EGFP positive cells (10.000 EGFP positive cells were counted). The MFIs of gate P3 were used for the diagram shown in Fig. 5A.

Table S1. Cloning primers

Table S2. Oligonucleotides for intron reporter vectors.

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