FIG 4.

GRA7 interacts with TRAF6 to modulate its ubiquitination. (A and B) THP-1 cells were transduced with lentivirus expressing nonspecific shRNA (shNS) or shRNA specific for TRAF6 with Polybrene (8 μg/ml). THP-1 (A) was transfected with an NF-κB reporter plasmid, and a pRL-TK Renilla plasmid was cotransfected for normalization. After 2 days, THP-1 cells were treated with rGRA7 (5 μg/ml) for 6 h and luciferase assays were performed (A) or for 18 h and culture supernatants were analyzed by cytokine ELISA (B). (C) RAW264.7 cells were stimulated with rGRA7 (5 μg/ml) for 20 min, and then immunoprecipitation (IP) with anti-TRAF6 (left) or anti-His (right) Ab and IB with anti-His, anti-Ub, or anti-TRAF6 were performed. Whole-cell lysates (WCLs)s were used for IB with anti-TRAF6 or anti-actin Ab. (D) Microscopy of BMDMs stimulated for 20 min with rGRA7 and then immunolabeled with Ab to TRAF6 (conjugated to the green fluorescent dye Alexa Fluor 488) or Ab to 6×His-GRA7 (conjugated to the red fluorescent dye tetramethyl rhodamine isocyanate). Scale bar, 5 μm. (E) Binding mapping. Schematic diagram of the structures of GRA7 and TRAF6. At 48 h posttransfection with mammalian GST or GST-GRA7 and truncated mutant constructs together with Flag-TRAF6 (left) or with GST or GST-GRA7 constructs together with Flag-TRAF6 and truncated mutant constructs (right), 293T cells were used for GST pulldown, followed by IB with anti-Flag Ab. WCLs were used for IB with anti-GST, anti-Flag, or anti-actin Ab. TM, transmembrane. (F) 293T cells were cotransfected with GST, GST-GRA7, or Flag-GRA7 together with HA-Ub and subjected to GST pulldown, followed by IB with anti-Flag or anti-HA Ab. WCLs were used for IB with anti-GST, anti-Flag, anti-HA, or anti-actin Ab. Data shown are the means ± SD of three experiments. Significant differences (**, P < 0.01; ***, P < 0.001) from control treated cultures are shown (A and B). The data are representative of three independent experiments with similar results (C to F).