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. 2015 Jul 29;35(4):e00233. doi: 10.1042/BSR20150091

Figure 2. ERK 1/2 expression increases CIITA transactivity leading to increased MHC II mRNA and surface expression.

Figure 2

(A) CIITA transactivation increases in the presence of expressed ERK1/2. Reporter assay: COS cells were transfected as indicated with MHC II HLA-DR-Luc reporter construct, Renilla, CIITA, CIITA S280A and ERK 1/2. Luciferase assays were performed in triplicate in three independent experiments, data are presented as fold increase in luciferase activity. Results are standardized to Renilla values and represent the mean ± S.D. *P<0.05. (B) Expression of MHC II mRNA is enhanced in the presence of expressed WT CIITA and ERK 1/2. mRNA assay: COS cells were transfected as indicated with Myc–CIITA, Myc–CIITA S280A, Flag–ERK1/2. Cells were harvested, RNA extracted and cDNA was prepared and quantified by real-time PCR. Levels of MHC II mRNA were normalized to GAPDH mRNA. Data presented are results of three independent experiments and represent ± S.E.M. *P<0.05, **P<0.005. Western blots indicate equal transfection and expression of Myc–CIITA and Flag–ERK1/2. (C) Expression of CIITA and ERK1/2 leads to an increase in cell surface expression of MHC II. Flow cytometry: COS cells were transfected with Myc-CIITA, Myc–CIITA S280A and Flag–ERK1/2. Seventy-two hours following transfection, cells were trypsinized, washed and incubated with PE-labelled anti-human HLA-DR antibody. Following incubation, cells were fixed and PE cell surface staining was measured by LSR Fortessa. (i) COS cells were transfected with Myc–CIITA (dark grey line) and COS cells were stained for isotype control (grey shaded). (ii) COS cells were transfected with Myc–CIITA (grey shaded) and were used to compare with Myc–CIITA and Flag–ERK1/2 (grey line). (iii) COS cells were transfected with Myc–CIITA (grey shaded) and were compared with cells transfected with CIITA S280A (grey dotted line). (iv) COS cells were transfected with Myc–CIITA (grey shaded) and compared with cells transfected with Myc–CIITA S280A and Flag–ERK1/2 (grey dotted line). Western blots indicate equal transfection and expression of Myc–CIITA, Myc–CIITA S280A and Flag–ERK1/2. Results shown are representative of three separate experiments.