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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: J Immunol. 2013 Jan 23;190(5):2415–2423. doi: 10.4049/jimmunol.1202535

Fig.2. Generation of IL-35 producing J558 and B16.F10 cells.

Fig.2

Mouse plasmacytoma J558 cells or B16F10 melanoma cells were co-transfected with an expression vector pORF9-IL-35 (Invivogen) and a selection vector (pCDNA-neo) or the control expression vector pORF9 (Invivogen) and pCDNA-neo. Stable cell lines that were resistant to G418 were generated. RT-PCR was used for detecting the expression of transcripts for recombinant IL-35, IL-12A, EBI3 and tumor antigen P1A in J558 cells (A) and IL-35, IL-12A and EBI3 transcripts in B16F10 cells (E). Immunofluorescence staining and ELISA revealed that IL-35 protein was produced by the generated J558 (B) and B16.F10 (F) cells. Flow cytometry was used for the analysis of MHC class I expression on the generated J558 cells (C) and B16 cells (G). MTT proliferation assay (MTT kit, ATCC) was used to measure growth and proliferation of J558 cells (D) and B16 cells (H). Bars indicate SD of triplicates.