Extended Data Figure 4: A20 proteins from wild-type, A20 OTU(C103A) cells, A20 ZnF4(C609A,C612A) cells, and A20 ZnF4(Y599A,F600A) cells are efficiently recruited to TNFR1 and regulate downstream signalling.

a, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 OTU(C103A) MEFs. b, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 ZnF4(C609A,C612A) MEFs. c, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 ZnF4(Y599A,F600A) MEFs. d, Immunoblot analysis Flag–TNF-treated whole-cell lysates in wild-type and in A20 OTU(C103A) MEFs. Asterisk, background band; arrow, phospho-MKK4. UnRx, untreated. e, Immunoblot analysis Flag–TNF-treated whole-cell lysates in wild-type and in A20 ZnF4(C609A,C612A) MEFs. Asterisk, background band; arrow, phospho-MKK4. UnRx, untreated. f, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 OTU(C103A) MEFs. Immunoblot analysis of whole-cell lysates from TNF-treated wild-type, A20 OTU(C103A), and A20 null MEFs following TNF pre-treatment to induce A20 expression, as well as TNF-treated A20 wild-type and A20 OTU(C103A) primary BMDMs, and TNF-treated A20 wild-type and A20 OTU(C103A) immortalized BMDMs all showed similar trends (not shown). g, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4 C609,612A E1A transformed MEFs. UnRx, untreated. Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4 C609,612A MEFs following TNF pre-treatment to induce A20 expression, and analysis of whole-cell lysates from TNF-treated A20 wild-type and A20 ZnF4 C609,612A primary BMDMs all showed similar trends (not shown). h, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4(Y599A,F600A) primary MEFs. For gel source data, see Supplementary Figs 6, 7. Data represent two to four biological replicates.