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. 2016 Oct;186(10):2679–2691. doi: 10.1016/j.ajpath.2016.06.014

Figure 4.

Figure 4

SOCS proteins are degraded less in T cells with dysfunctional neddylation pathway. Protein analysis by Western blot of p65 isoform of NF-κB protein using nuclear (N) and cytosolic (C) fractions from wild-type (WT) and knockout (KO) T cells (A) or WT T cells cultured in the presence or absence of vehicle or 500 nmol/L MLN4924 (B), subjected to or withheld from concurrent stimulation with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28. Plots below Western blot images show densitometric and statistical analysis of p65 NF-κB presence in the nuclear fraction and cytosolic fraction. Lamin A/C and lactate dehydrogenase proteins were analyzed to demonstrate the presence of nuclear and cytosolic fractions, respectively. One representative experiment of three is shown. C: Immunocytochemistry analysis of p65 NF-κB (column 3) localization in bone marrow–derived dendritic cells cultured in the presence or absence of MLN4924 and stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28. Cell nuclei were stained using DAPI (column 1). Cytoplasmic actin was stained with phalloidin (column 2). Merged images of staining (column 4). One representative experiment of three is shown. D: Gene expression in WT and KO T cells identified in Affymetrix microarray analysis. The data set and top 24 enriched genes shown were obtained from two biological replicates. E: SOCS1 and SOCS3 gene expression in CD90.2+ T cells, stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28 where indicated, and treated with 500 nmol/L MLN4924 or vehicle. Flow cytometric analysis of SOCS1 protein (F) and SOCS3 protein levels (G) in WT and KO C57BL/6 T cells stimulated with irradiated (30 Gy) allogeneic BALB/c splenocytes for indicated time. One representative experiment of two is shown in groups treated in triplicate. H: Western blot analysis of SOCS1 protein levels in WT T cells treated with MLN4924 500 nmol/L and stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28 for 72 hours. One representative experiment of three is shown. P < 0.05, ∗∗∗P < 0.001. Original magninfication, ×60 (C). SLCK, SAGfl/fl × Lck-Crel; Stim, stimulated; Unstim, unstimulated.