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Figure 4 | BMC Immunology

Figure 4

From: ER stress affects processing of MHC class I-associated peptides

Figure 4

ER stress impairs cell surface MHC I expression through posttranscriptional mechanism(s). EL4 cells were incubated in DMEM control medium containing glucose (4.5 mg/ml), or in medium lacking glucose or supplemented with 0.25 mM of palmitate for 18 hours. (A) ER stress does not decrease MHC I mRNA levels. H2Kb, H2Db and β2m mRNA levels were assessed and analyzed by RT-qPCR. Expression levels were normalized to the endogenous control gene β-actin. Transcript levels of glucose-starved (green) or palmitate-treated (orange) cells were compared with basal mRNA values of control cells (dotted line), which were set to 1. Bars represent the mean and SD from three independent experiments performed in triplicate. No significant differences were detected between untreated and treated cells (P < 0.05). (B) ER stress does not affect total MHC I protein amount. MHC I proteins from whole cell lysates were detected by Western blot with anti-MHC I antibodies. α-tubulin was used as loading control. A representative image of three independent experiments is shown. (C) ER stress differentially affects synthesis of MHC I. EL4 cells were incubated in control conditions, deprived of glucose or treated with 0.25 mM of palmitate for 17 hours and pulse-labeled with [35S]methionine/[35S]cysteine for 1 hour. Cell extracts were lysed and subjected to immunoprecipitation with anti-MHC I antibody or the corresponding isotypic antibody. Immunoprecipitated proteins were separated by SDS-PAGE and analyzed by fluorography. One representative experiment out of two is shown.

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