Fig 1: The PRY-SPRY domain of TRIM41 directly binds the CARD domain of BCL10. a Wild type BMDM were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h as indicated. Then whole cell extracts (WCE) were immunoprecipitated with anti-TRIM41 antibody plus protein A/G beads. Components in the TRIM41 complex were examined by Western blotting. b 1 μg GST or GST-TRIM41 was coincubated with 1 μg recombinant CARD9, CARD11, or BCL10 for 1 h. Then sepharose 4B beads were used to pull down the GST complex. Components pulled down by the beads were examined by Western blotting. c Schematic illustration of domains in full-length (FL) or fragments (F) of TRIM41-Flag or BCL10-Myc. R, ring finger domain; B, B-box domain; CC, coiled-coil domain; CTD, C-terminal domain. Numerical numbers indicate the site of amino acids. d, e HEK293T cells were transiently transfected with Flag-tagged TRIM41 (or mutants) and Myc-tagged BCL10 (or mutants) as indicated for 48 h. Whole cell extracts (WCE) were immunoprecipitated with anti-Flag Sepharose Beads (d) or anti-Myc Sepharose Beads (e), and the associated BCL10 or TRIM41 was examined by Western blotting. f, g RAW264.7 cells grown on cover slides were transiently transfected with green fluorescent protein (GFP)-tagged TRIM41 and red fluorescent protein (RFP)-tagged BCL10 for 48 h, and then infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h. After counterstained with DAPI, cells were examined by confocal microscope (f; Scale bar, 100 nm). Ten double-positive randomly-selected cells on cover slides were measured for fluorescence colocalization, and the data are presented as mean ± SD (g)
Fig 2: TRIM41 mediates Lys63-linked polyubiquitination of BCL10. a-c Trim41+/+ or Trim41–/– BMDM were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h. Then whole-cell extracts (WCE) heated in buffer containing 1% SDS were immunoprecipitated (IP) with anti-BCL10 (a), anti-CARD9 (b), or anti-CARD11 (c) antibody plus protein A/G beads. Polyubiquitination of BCL10 (a), CARD9 (b), and CARD11 (c) was examined by Western blotting. d HEK293T cells were transiently transfected with Flag-tagged TRIM41 (or mutant) and Myc-tagged BCL10 vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. e HEK293T cells were transiently transfected with Flag-tagged TRIM41, Myc-tagged BCL10, and HA-tagged Ub (mutants) vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. f After incubation for 30 min, the in vitro polyubiquitination system was boiled for 5 min, and then polyubiquitinated BCL10 was examined by Western blotting against Ub after immunoprecipitations. g HEK293T cells were transiently transfected with Flag-tagged TRIM41 and Myc-tagged BCL10 (mutant) vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. One representative experiment of three is shown
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