Fig 1: TRAF6 E3 ligase activity is required for the IL-1ß-dependent activation of the TAB1–TAK1 heterodimer in TAB2/3 DKO cells.(A) TAB2/3 DKO (clone 4 from Figure 1A) and TAB2/TAB3/TRAF6 TKO IL-1R* cells were stimulated with IL-1ß for the times indicated, and cell extracts subjected to SDS–PAGE and immunoblotting with the antibodies used in Figure 1 and an antibody recognizing IRAK4 phosphorylated at Thr245 and Ser346. (B) As in A, except that WT TRAF6 or the indicated E3-ligase-inactive mutants of TRAF6 were re-expressed in the TAB2/TAB3/TRAF6 TKO IL-1R* cells. (C) WT and TAB2/3 DKO cells were incubated for 72 h with siRNA against Ubc13 and stimulated for 10 min with 5 ng/ml IL-1ß. Other details are as in A.
Fig 2: TAK1[D175A] × Vav-iCre mice have enlarged lymphoid organs, increased B cell and neutrophil numbers and normal expression of TAK1 and TAB proteins.(A) Representative image of spleens of 12 week old WT and TAK1[D175A] × Vav-iCre mice (D175A), scale bar = 1 cm. (B) Spleen weights of 12 week old WT (n = 4) and TAK1[D175A] × Vav-iCre mice (n = 4). Each symbol represents an individual mouse. (C–E) As in (B) except that splenocyte (C), B cell (D) and neutrophil (E) numbers in the spleen are shown. (F) Representative images of axillary (upper panel) and inguinal (lower panel) lymph nodes (LN) of 12 week old WT and TAK1[D175A] × Vav-iCre mice (D175A). (G) Total cell numbers in axillary and inguinal LN of 12 week old WT (n = 4) and TAK1[D175A] × Vav-iCre mice (n = 4). (H) As in (G), except B cell numbers in the LN are shown. Statistical significance between the two genotypes was calculated using the unpaired t-test with Welch's correction; * denotes P < 0.05. (I) Extracts of BMDM (10 µg protein) from three separate WT mice or three separate TAK1[D175A] × Vav-iCre mice (D175A) (+,+,+) were denatured in SDS, subjected to SDS–PAGE, transferred to PVDF membranes and immunoblotted using the ECL detection system (GE Healthcare) with antibodies recognising TAK1, TAB1, TAB2, TAB3 and GAPDH as a loading control.
Fig 3: IL-1ß signaling in TAB1 KO IL-1R* cells.(A) Generation of two clones of TAB1 KO IL-1R* cells. Other details are as in Figure 1A. (B) WT IL-1R* cells and TAB1 KO cells (clone 44) were stimulated for up to 2 h with IL-1ß . Cell extracts were denatured in SDS, subjected to SDS–PAGE and immunoblotted with the antibodies indicated. (C–E) As in B, except that RNA was extracted from the cells at the times indicated and the formation of I?Ba (C), A20 (D) and IL-8 (E) mRNA was measured by qRT-PCR relative to 18S ribosomal mRNA and normalized to the level found in WT cells stimulated with IL-1ß. The results are presented as arithmetic mean ± SEM for three independent experiments, each performed in triplicate. (F) As in E, except that IL-8 secreted into the culture medium was measured by ELISA.
Fig 4: IL-1 signaling is restored to TAB1/2/3 TKO cells by the re-expression of TAB1.(A) The experiment was carried out as in Figure 3A, except that TAB1/2/3 TKO cells (clone A4 from Figure 1A) and WT IL-1R* cells were used. (B) As in A, except that HA-TAB1 was re-expressed in the TAB1/2/3 TKO IL-1R* cells where indicated and the cells stimulated for 10 min with 5 ng/ml IL-1ß. Extracts from WT cells (20 µg of protein) and TAB1/2/3 TKO IL-1R* cells (40 µg of protein) were then processed as in A (top five panels). In the bottom two panels, TAK1 was immunoprecipitated from the extracts and immunoblotted with anti-TAB1 and anti-TAK1 to confirm that the re-expressed TAB1 had recombined with TAK1.
Fig 5: IL-1 signaling in different TAB- and TRAF6-deficient cells.(A) Generation of TAB1/TRAF6 DKO cells from TAB1 KO cells (clone 44 from Figure 5A). (B) WT TRAF6 and the E3 ligase-inactive TRAF6[L74H] and TRAF6[120-522] mutants were re-expressed in TAB1/TRAF6 DKO cells (clone 1 from A). These cells, TAB1 KO cells (clone 44 from Figure 5A) and TAB1/TRAF6 DKO cells not re-transfected with TRAF6, were then stimulated with IL-1ß for the times indicated. Other details are as in A. (C) HA-TAB2 or the K63-Ub-binding-defective HA-TAB2[T674/F675A] mutant were re-expressed in TAB1/2/3 TKO IL-1R* cells (clone A4 from Figure 1A). These cells, TAB1/2/3 TKO cells not re-transfected with HA-TAB2 and WT IL-1R* cells, were stimulated for 10 min with 5 ng/ml IL-1ß. Cell extracts (20 µg of protein-WT cells or 40 µg of protein-TAB1/2/3 TKO cells) were subjected to SDS–PAGE and immunoblotted with the antibodies indicated.
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