TRIM21 differentially regulates the stability of IRF5 isoforms.
A–D, top left, HEK-TLR7 cells were transfected with Myc-tagged IRF5 isoforms (A, V1; B, V2; C, V3; D, V5) in presence or absence of Xpress-TRIM21. The day after transfection cells were treated with cycloheximide (100 µg/ml) in combination with CL097 (5 µg/ml) for the indicated times. Levels of IRF5, TRIM21 and α-Actinin were determined by immunoblot and levels of IRF5 normalized to α-Actinin were calculated and plotted, *p<0.05. A–D, top right, HEK293T were transfected with plasmids encoding the luciferase reporter gene under the control of the IFNA4 promoter, Myc-tagged IRF5 isoforms and MyD88 in presence or absence of Xpress-TRIM21. The TK-Renilla plasmid was used as internal control. Luciferase activity was measured 48 hours after transfection and normalized to renilla activity. Results are shown as fold activation over Empty Vector control, *p<0.05. A–D, bottom, HeLa cells were transfected with 1 µg of plasmids encoding GFP-tagged IRF5 (green) and mRFP-TRIM21 (red) and left untreated or stimulated with Imiquimod for 3 hours. Cells were fixed mounted in DAPI in order to visualize nuclei (blue) and images were taken under oil immersion at 63× magnification. Images shown are from a single experiment and are representative of three independent experiments.