Protein expression and localization of mutant NF-κB2 in transfected cells. (A) HEK293T cells were transiently transfected with the indicated constructs in the presence and absence of NIK coexpression. Cell lysates were analyzed for phosphorylation of p100 and for p100, p52, and mutant expression by western blotting. E418X mutant size was closed to the active form p52. All other mutants are failed phosphorylation and processing to p52 in the presence of NIK expression. Molecular weight markers are indicated on the left size (kilodaltons). (B) HEK293T cells were transiently transfected indicated WT or mutant vector (pcDNA3-HA) with or without Flag-NIK. Nucleus was labeled with DAPI (blue). Original magnification ×175. Cells were stained with anti-Flag and anti-HA antibody, followed by Alexa 488-conjugated (for HA-NF-kB2, green) and Alexa 568-conjugated (for Flag-NIK, red) secondary antibody. Data shown are representative of 3 experiments.