Figure 3

Stabilization of pHLA complexes by single non-canonical short peptides. (a) Stabilization of pHLA complexes formed by HLA-A*11:01 heavy chain and β2 m with HLA-A*11-restricted SSCSSCPLSK or its C-terminal truncated peptides (SCSSCPLSK, CSSCPLSK and SSCPLSK) in a dose-dependent manner (0, 0.1, 1 and 10 mg/ml) as determined by native western blot with W6/32 antibody. pHLA complex of HLA-A*11:01 heavy chain and β2 m refolded with 10 mg/ml of full-length SSCSSCPLSK peptide served as the positive control (Pos). HLA-A*11:01 heavy chain and HLA-A*11:01 heavy chain with β2 m served as the negative controls. (b) Tabulation of MHC class I molecules refolding in-vitro with N-terminal (N-term) 7mer peptide (P2 anchor underlined) and/or C-terminal (C-term) 7mer peptide (PΩ anchor underlined) as determined by native western blot with W6/32 antibody shown in the lower panels. Presence of bands detected by W6/32 antibody in the native western blot indicates the presence of stabilized pHLA complexes. Due to the difference in overall charge-to-mass ratio of the different pHLA complexes formed by different peptides and peptide lengths, a variation in the migration rates of the different pHLA complexes in the native PAGE gels can be observed. For each indicated HLA allele, heavy chain (H), heavy and β2 m light chain (HL) served as the negative controls while pHLA complex with full peptide (FL) served as the positive control for each western blot panel. EBV, Epstein-Barr Virus; HBV, Hepatitis B Virus; MTB, Mycobacterium tuberculosis.