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. 2015 Jan 2;125(2):593–606. doi: 10.1172/JCI77780

Figure 2. HLA-DR expression and overexpression of mutated and wild-type IDH1 in glioma cell line LN229 as analyzed by PLA, a tool for analysis of MHC class II–peptide interaction.

Figure 2

(A) PLA scheme using anti–HLA-DRA and H09 primary antibodies. Red represents rolling circle amplification, and α and β represent HLA-DR chains. pIDH, IDH1 epitopic peptide. (B) Flow cytometry and IF (IDH1D252G R132H) of glioma cell line LN229. Green, HLA-DR. Data are representative of 3 experiments. DG RH, IDH1D252G R132H; DG, IDH1D252G; iso, isotype control; specific, HLA-DRA–specific antibody. Scale bar: 20 μm. (C) Scheme for enzymatic activity of IDH1 mutants. α-KG, α-ketoglutarate. (D) R-2-HG measurement in IDH1D252G R132H, IDH1D252G, IDH1R132H (RH), and IDH1WT (WT) LN229 cells by enzymatic assay. Data are representative of 3 experiments. (E) Western blot and (F) and IF of LN229 cells overexpressing IDH1D252G or IDH1D252G R132H detecting IDH1. Tubulin was used as loading control. Green, IDH1R132H; blue, DAPI. Data are representative of 3 experiments. EV, empty vector. Scale bar: 20 μm.