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. 2013 Oct 4;8(10):e75200. doi: 10.1371/journal.pone.0075200

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© 2013 Friend et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Representative immunoblots and (B) quantification of NEDD8 relative density from MA5.8ζ cells stimulated with 5 µg/mL α-CD3 with or without 200 nM MLN4924 (n = 4); P<0.05 compared to 0 min with MLN4924. (C) Representative immunoblots and (D) quantification of NEDD8 relative density from primary CD4⁺ T cells stimulated with 5 µg/mL α-CD3 or incubated with either 3 µM MLN4924 for 2 h as a deneddylated control or 2 mM OPT for 30 min as a neddylated control (n = 4). (E) Representative cullin immunoblots from primary CD4⁺ T cells stimulated as in (C) (arrows indicate neddylated CULs) (n = 3). (F) Representative immunoblots and (G) quantification of NEDD8 relative density from human cord blood CD4⁺ T cells stimulated with 10 µg/mL α-CD3 and 2 µg/mL α-CD28 or incubated either with 3 µM MLN4924 for 2 h or 2 mM OPT for 30 min (n = 4). Values represent mean ± s.e.m.; *P<0.05, **P<0.01, ***P<0.001 compared to 0 min.

Inline graphic — (A) Representative immunoblots and (B) quantification of NEDD8 relative density from MA5.8ζ cells stimulated with 5 µg/mL α-CD3 with or without 200 nM MLN4924 (n = 4); P<0.05 compared to 0 min with MLN4924. (C) Representative immunoblots and (D) quantification of NEDD8 relative density from primary CD4⁺ T cells stimulated with 5 µg/mL α-CD3 or incubated with either 3 µM MLN4924 for 2 h as a deneddylated control or 2 mM OPT for 30 min as a neddylated control (n = 4). (E) Representative cullin immunoblots from primary CD4⁺ T cells stimulated as in (C) (arrows indicate neddylated CULs) (n = 3). (F) Representative immunoblots and (G) quantification of NEDD8 relative density from human cord blood CD4⁺ T cells stimulated with 10 µg/mL α-CD3 and 2 µg/mL α-CD28 or incubated either with 3 µM MLN4924 for 2 h or 2 mM OPT for 30 min (n = 4). Values represent mean ± s.e.m.; *P<0.05, **P<0.01, ***P<0.001 compared to 0 min.