Supplementary Figure 9: Analysis of allele effects on A20 phosphorylation and control of NF-κB.
From: Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity
(a) A20 accumulation in βTC3 mouse insulinoma cells transfected with wild-type (WT) A20, C103A A20, T108A;I207L A20, I325N A20, C243Y A20, S381A A20 or S184N A20 was determined by densitometry analysis of independent experimental immunoblots. Fast migratory (A20) 98 kDa A20 was corrected to loading control and compared to WT A20 of the same immunoblot [(FM A20/β-actin)/A20]. (b) Results of mass spectrometry analysis of full length WT A20, C103A mutant A20, or I325N mutant A20 expressed in and purified from HEK293 cells and analysed by mass spectrometry for phosphorylation of the indicated residues as per references18,19. Data representative to two independent experiments. (c) βTC3 cells co-transfected with an NF-κB.luciferase reporter and a CMV.βgal expression construct alone or with PCDNA3.1 encoding human wild-type A20 (hA20 WT; blue), A20 T108A/I207L (purple), A20 C243Y (orange), A20 S184N (grey), A20 I325N (red) or A20 S381A (white). Cells were stimulated with (+) 200 U/ml hTNF for 8 h or left untreated (-). Data presented as relative luciferase units. Each column represents 3-5 independently transfected aliquots of cells within one experiment marked by circle symbols (representative of 3 independent experiments) and columns are arithmetic means ± s.e.m. Statistical comparison by two-tailed Student’s t-test against WT hA20 (blue). (d) βTC3 cells co-transfected with a NF-κB.luciferase reporter and a CMV.βgal expression construct alone or with PCDNA3.1 encoding human wild-type A20 (hA20 WT; blue), or A20 I325N (I325N; red), or A20 with serine 381 substituted to non-phosphorylatable alanine (S381A or I325N S381A), or to phosphomimetic glutamate (S381E or I325N;S381E). Cells were stimulated with (+) 200 U/ml hTNF for 8 h or left untreated (-). Data presented as relative luciferase units. Each column represents mean of 3-6 independently transfected aliquots of cells within one experiment marked by circle symbols (representative of 3 independent experiments) and columns are arithmetic means ± s.e.m. Statistical comparison by two-tailed Student’s t-test against stimulated (*) or unstimulated (^) WT hA20 (blue). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
