Siinfekl

To exchange the native peptide with SIINFEKL (Peptron, Daejeon, Korea), 5 mg of K^b-attached NPs was incubated with 20 μM or 100 μM SIINFEKL for 18 h at 37°C, washed twice with PBS, and resuspended in 0.5 ml of PBS. The level of SIINFEKL bound to the K^b-attached NPs was analyzed with mAbs recognizing the SIINFEKL-K^b complex (25-D1.16; BioLegend).

Soluble OVA gene, which was amplified from pCI-neo-sOVA (a gift from Maria Castro, Addgene plasmid 25 09818 (link)), and monomeric enhanced green fluorescent protein (mEGFP) gene, which was amplified from mEGFP-N1 (a kind gift from Michael Davidson, Addgene plasmid 54767), were genetically fused via P2A translational skipping sequence and were cloned in the Sleeping Beauty transposon plasmid with the human elongation factor 1α promoter.19 (link) This plasmid, pT2-EF-OVA-mEGFP, was electroporated together with the Sleeping Beauty Transposase plasmid, pCMV(CAT)T7-SB100 (a gift from Zsuzsanna Izsvak; Addgene plasmid #34 87920 (link)), into TRAMP-C2 by Nucleofector 4D instrument. The electroporated cells were kept in maintenance medium (DMEM supplemented with 10% FBS, 0.005 mg/mL bovine insulin, 1 nM DHT and cell sorted based on mEGFP expression using FACS Aria I cell sorter. The expression of OVA on sorted cells were confirmed by western blotting using rabbit polyclonal OVA antibody (ab186717) at 1:4000 dilution and flow cytometry using PE anti-mouse H-2K^b bound to SIINFEKL (BioLegend) before incubating with or without MCTP-39 for 48 hours. The expression of OVA was analyzed after 48 hours by flow cytometry using BDLSRIIA cytometer, and data were analyzed by FCS Express V.7 Research Edition.