IMiD3 induces CD28 phosphorylation and NF-κB activation. CD3 T cells negatively selected from healthy donors were incubated for different time points in the presence or absence of IMiD3 (10 μM). (A) Cell lysates were immunoprecipitated with antihuman CD28 Ab. Immune complexes were resolved on SDS-PAGE, transferred on polyvinylidene fluoride (PVDF) membrane, and analyzed by blotting with antiphosphotyrosine Ab showing a 44-kDa band representing tyrosine phosphorylation. DMSO and anti-CD28 Ab were used as negative and positive controls, respectively. (B) Nuclear extracts were collected and prepared for EMSA as described previously.¹²  DMSO and TNF-α (5 ng/mL) were used as negative and positive controls, respectively. Densitometric measurement with data normalized to TNF-α as 100% showed 62.5%, 59%, and 90% activation at 30 minutes, 60 minutes, and 3 hours, respectively, following IMiD3 treatment with 64% activation for negative control.