Figure 4: Pellino regulates MyD88 ubiquitination and turnover through K-48 linkage.

(a,b) S2 cells were transfected with combinations of expression plasmids as indicated. Forty-eight hours after transfection, cell lysates were prepared, immunoprecipitated with anti-Flag beads (a) or anti-Myc beads (b), followed by immunoblot analysis with the indicated antibodies. Expression levels of transfected proteins in whole-cell lysates are shown in bottom panel. (c) S2 cells were treated with the dsRNAs as indicated for 48 h, and then transfected with combinations of expression plasmids as indicated. Forty-eight hours after transfection, cell lysates were prepared, immunoprecipitated with anti-Flag beads, followed by immunoblot analysis with the indicated antibodies. Expression levels of transfected proteins in whole-cell lysates are shown in bottom. (d,e) S2 cells were transfected with combinations of expression plasmids as indicated (d) or cells were also pretreated with pellino dsRNA (e). Cell lysates were then used to perform immunoprecipitation experiments with anti-Myc beads, followed by immunoblot analysis with anti-HA antibodies to show ubiquitination pattern of MyD88. (f,g) Similar to d and e, except the DNA vector expressing HA-Ub-K48 was used to examine the MyD88 K48-linked ubiquitination pattern under overexpression (f) or knockdown (g) of pellino condition. (h) S2 cells were transfected with expression constructs as indicated, and after 48 h post transfection, cells were treated with CHX (50 ng ml⁻¹) for different times, followed by immunoblotting to check MyD88 expression levels. Densitometry analysis to quantify MyD88 expression is shown in the bottom panel. Error bars represent s.d. (n=3). (i) S2 cells were treated with dsRNAs targeting gfp or pellino for 48 h, then transfected with Myc-MyD88. After 48 h, cells were treated with CHX (50 ng ml⁻¹) for different times, followed by immunoblotting to check MyD88 expression levels. Densitometry analysis to quantify MyD88 expression is shown in the bottom panel. Error bars represent s.d. (n=3). For data from h and i, the Log-rank test was used to analyse the variance of the protein stability between two groups. ***P<0.001, versus control groups.