Defective translocation of p65 and p50 in SV40-fibroblasts and in IKKα-deficient cellular models. (A) Western blot of cytoplasmic and nuclear protein extracts from SV40-fibroblasts activated with TNFα for 30 min assessing the translocation of p65/RELA and p50 in the nucleus. α-Tubulin was used as a loading control of cytoplasmic extract while Lamin A/C was used for nuclear extracts. (B) Quantification of nuclear translocation for p65/RELA and p50. Statistical analysis was performed using an unpaired t test (**P < 0.01). Representative of at least three independent experiments. (C) Western blot of cytoplasmic and nuclear extracts from HEK293T cells (IKKα ΚΟ or not as indicated) after 30 min of TNFα (10 ng/ml) stimulation. RELA/p65 and p50 were revealed using a specific antibody to assess for translocation in the nucleus. α-Tubulin was used the loading control of cytoplasmic extract while Lamin A/C was used for nuclear extracts. IκΒα was probed as a control of stimulation. (D) Barplots show the quantification of translocation normalized to the quantity of Lamin A/C for RELA/p65 and p50 after stimulation. An unpaired t test was used to perform statistical analysis (*P < 0.05). (E) Western blot of cytoplasmic and nuclear extracts from 3T3-NIH IKKα knocked-out transduced back with a lentivirus coding for the human IKKα (or empty) with or without indicated mutations (p.K44A, kinase dead, p.H142R, patient variant). Cells were subjected to mouse TNFα stimulation (30 min, 10 ng/ml) before protein extraction. RELA/p65 was assessed as well as α-tubulin (used as a loading control of cytoplasmic extracts) and Lamin A/C (used as a loading control for nuclear extracts). (F) Barplots show the quantification of p65 translocation after stimulation for each transduced cell line. All figures are representative of at least two independent experiments. Source data are available for this figure: SourceData F8.