Fig. 2: MGF 505–3R interacts with MyD88 and promotes its degradation via K48-linked ubiquitination.

A HeLa cells were transfected with pcDNA3.1–Flag–MyD88, empty vector, or pCMV–myc–MGF 505–3R for 24 h. Flag–MyD88 expression was detected by Western blotting, with β-actin used as a loading control. B HeLa cells were transfected with either an empty vector or pCMV–myc–MGF 505–3R for 24 h. Endogenous MyD88 levels were assessed by Western blotting, with GAPDH as a loading control. C HEK 293T cells were transfected with pcDNA3.1–Flag–MyD88, an empty vector, or pCMV–myc–MGF 505–3R for 24 h. Whole cell lysates (WCL) were subjected to immunoprecipitation using anti-Flag or anti-Myc antibodies. The resulting immunoprecipitates were analyzed by Western blotting with the specified antibodies to detect protein–protein interactions. D HeLa cells were transfected with pGL4.32–NF–κB–Luc, along with either an empty vector, pcDNA3.1–Flag–MyD88 or pCMV–myc–MGF 505–3R for 24 h, followed by treatment with inhibitors MG132 (10 μM), BafA1 (10 μM), or 3–MA (25 nM) for 6 h. Luciferase activity was then measured to determine the effect on NF–κB signaling (mean ± S.D., n = 4, **p < 0.01). E, F HEK 293T cells were transfected with pcDNA3.1–Flag–MyD88, empty vector, or pCMV–myc–MGF 505–3R for 24 h. Whole cell lysates (WCL) were immunoprecipitated with anti-Flag, anti-Myc, anti-ubiquitination, anti-k48, or anti-k63 antibodies, and the immunoprecipitates were analyzed by Western blotting using the indicated antibodies to detect protein interactions.