Figure 7

Increased presentation of SIINFEKL peptide derived from ER-localized relative to cytosolic HEL protein during ER stress. (A) ER stress differentially affects surface expression of K^b-SIINFEKL complexes. EL4 stable cell lines were incubated in medium lacking glucose or containing low glucose (1 mg/ml) or high glucose (4.5 mg/ml) or supplemented with palmitate (2.5 mM) for 18 hours. K^b-SIINFEKL abundance was assessed with the 25-D1.16 monoclonal antibody and APC-conjugated anti-mouse IgG₁ antibody. Graph represents MFI values of glucose-deprived EL4/HEL-Cyto-SIINFEKL (blue) or EL4/HEL-ER-SIINFEKL (red) cells normalized to values of control cells, which were set to 1 (dotted line). (B) ER stress differentially affects surface expression of newly generated K^b-SIINFEKL complexes. EL4 stable cell lines were incubated under control conditions (top), deprived of glucose (middle) or treated with 0.25 mM of palmitate (bottom) for 18 hours. Existent MHC-I complexes were eluted by acid strip and expression of new K^b-SIINFEKL complexes was assessed as in A at the indicated times. MFI values of unstripped cells incubated under normal conditions and representing normal level of K^b-SIINFEKL in each cell line were used to normalize MFI values of stripped cells. Bars represent the mean and SD from three independent experiments performed in triplicate. *P< 0.05 and **P < 0.01 when comparing normalized K^b-SIINFEKL expression in EL4/HEL-Cyto-SIINFEKL with that of EL4/HEL-ER-SIINFEKL.