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. 2022 Jul 8;8(27):eabq0084. doi: 10.1126/sciadv.abq0084

Fig. 5. LYN phosphorylates a conserved tyrosine in IRFs controlling K48 ubiquitination and stability.

Fig. 5.

(A) Sequence alignment of MyD88-induced IRFs. The conserved tyrosine is indicated by arrow. (B) In vitro LYN kinase assay using recombinant proteins expressed in HEK293T cells. Incorporation of P32 was analyzed by phosphor imaging. (C) IP/IB experiments using HEK293T cells and transfected proteins. (D to F) IP/IB experiments using HEK293T cells and transfected proteins. (G) Nuclear translocation assay of wt and SPOP-deficient RAW264.7 cells that were reconstituted retrovirally with IRF5 wt or IRF5 Y118F. (H) IP/IB analysis of IRF5 phosphorylation using cells described in (G). MG, MG-132. (I) IP/IB analysis of K48 ubiquitination of IRF5 in wt and SPOP-deficient RAW264.7 cells. (J) IP/IB analysis of K48 ubiquitination of IRF5 (I) using transfected HEK293T cells. (K) IB analysis of IRF5 and phospho-LYN upon overexpression of LYN wt or kinase-dead LYN [LYN-KD (D385G)]. (L) IB analysis of HEK293T cells that were transfected with IRF5 wt or IRF5 Y118 (L), followed by cycloheximide (CHX) treatment to block protein translation for indicated time points.