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. Author manuscript; available in PMC: 2016 Sep 15.
Published in final edited form as: Immunity. 2015 Aug 25;43(3):463–474. doi: 10.1016/j.immuni.2015.07.022

Figure 3. Immunological and biochemical consequences of OST dysregulation.

Figure 3

(A) Quantitative RT-PCR array analysis of immune gene activation in wild type BMDMs treated with indicated glycans. Free glycans from WT and Trex1−/− MEFs were untreated or treated with a cocktail of DNase, RNase and protease before being added to BMDMs for 20 h. Each data point represents one gene as in Figure 1F. (B–C) Quantitative RT-PCR analysis of immune gene activation in wild type BMDMs treated with indicated glycans. Glycan source in B is free glycans isolated from Trex1−/− MEFs (pooled glycans) or from FACE gels (gel-purified glycans). Glycan source in C is N-glycan isolated from increasing amount of RNase B protein (Sigma Cat#R1153, PNGase digest) or from buffer alone. Ifit1 and Cxcl10 mRNA were analyzed by qRT-PCR. Right, FACE analysis of glycans used in B and C. (D) ‘In-cell’ N-glycosylation assay. WT and Trex1−/− MEFs were permeabilized by SLO and incubated with reaction buffer (see Method) and control or acceptor peptide. Only the acceptor peptide contains one N-glycysolation site. Transferred N-glycans were then cleaved by PNGase and analyzed by FACE (top gel). Free glycans were also analyzed from the same experiment (bottom gel). Free glycans quantified in this assay are from LLO hydrolysis during the assay, not the pre-existing free glycans in the cell (much smaller in size). Data are representative of at least two independent experiments. Error bars, SEM (same throughout). Mann-Whitney test (A). Unpaired t-test (B). See also Figure S3.