Fig. 4: CIS (Cytokine inducible SH2 protein) acts as a substrate receptor for CRL5.

a IBAQ intensities for CIS and SOCS2 proteins. CD4⁺ T cells from 8–10-week-old mice were restimulated for 4 h with either anti-CD3 and anti-CD28, or anti-CD3 and anti-CD28 in the presence of IL-4, or anti-CD3 and anti-CD28 in the presence of IL-4 and treated with Neddylation inhibitor (NAEi) for the final one hour. After the respective treatment, the lysates were immunoprecipitated by anti-Cul5 or IgG antibodies, and samples were analyzed by mass spectrometry. n = 2 biologic replicates of cells examined in two independent experiments. b Immunoblot showing interaction of Cul5 and CIS in primary CD4⁺ T-cells. n = 2 biologic replicates of cells examined in two independent experiments. Representative Immunoblot (c) and fold change in CIS expression (d) in D10 cells. n = 3 biologic replicates of cells examined in three independent experiments. To calculate fold change, the normalized CIS level at 0 h was adjusted to 1 and the relative change in the expression of CIS protein was calculated accordingly. e Table showing components of the CRL5 E3 ubiquitin complex that were identified by CIS IP and IgG IP in D10 cells. Cells were stimulated in the presence of IL-4 and NAEi for 4 h. Total number of peptides identified in IP is listed in the table. n = 2 biologic replicates of cells examined in two independent experiments. f Immunoblot of Cul5 IP showing its interaction with CIS in D10 cells. n = 3 biologic replicates of cells examined in three independent experiments. Immunoblot (g) and fold change (h) of CIS in D10 cells stimulated in the presence of exogenous IL-4 or anti-IL-4 for 4 h. n = 3 biologic replicates of cells examined in three independent experiments. Immunoblot (i) and fold change of CIS protein (j) in murine CD4⁺ T-cells isolated from 8–10-week-old mice. n = 3 biologic replicates of cells examined in three independent experiments. k Immunoblot showing expression of CIS protein in murine CD4⁺ T cells. n = 3 biologic replicates of cells examined in three independent experiments. Data is presented as Mean ± SEM in panels d, h and j. p-value was calculated using unpaired two tailed t-test. Source data are provided as Source Data file.