Fig. 1: Cul5 expression and activation increased following CD4⁺ T cell stimulation.

Immunoblot analysis showing neddylated and unneddylated forms of Cul5 in CD4⁺ T cells. a CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 for 48 h after which cells were washed and kept in IL-2 containing media referred as “Resting media”. Resting murine CD4⁺ T cells were either “stimulated” with anti-CD3 and anti-CD28 for 4hrs or kept in IL-2 containing “resting” media. Nedd8 inhibitor (MLN4924) (NAEi, 1 μM) was added during the final 1 h of restimulation. n = 3 biologic replicates were examined in three independent experiments. b Naive CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time, after 72 h cells were transferred to fresh IL-2 containing media n = 3 biologic replicates were examined in three independent experiments. c Schematic of Cul5 gene with loxP insertions. d Immunoblot showing expression of Cul5 in CD4⁺ T cells isolated from spleens of control and Cul5^fl/flCD4-Cre mice. n = 3 biologic replicates were examined in three independent experiments. e Representative flow plots showing the frequencies of CD4⁺ and CD8⁺ T cells in each thymus. f Bar graphs show the compiled data for the frequencies of CD4⁺ and CD8⁺ T cells in thymi. n = 6 biologic replicates for each genotype were examined in two independent experiments. g Representative flow plots showing the frequencies of CD62L⁺ (Naïve) and CD44⁺CD4⁺ T cells from spleens, lymph nodes, and lungs of Cul5^fl/flCD4-Cre and control mice. h Compiled data showing the frequencies of CD44⁺CD4⁺ cells from lungs of Cul5^fl/flCD4-Cre and control mice. n = 20 WT and n = 22 Cul5^fl/flCD4-Cre animals were examined in seven independent experiments. Data in panels f, h are presented as Mean ±  SEM. p-values were calculated using an unpaired two-tailed t-test. Source data are provided in the Source Data file.