11259596

  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (5 min)
    *** cells: HEK293 in BMS
    *** source: 11259596-Fig-4
  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (5 min)
    *** cells: HEK293I1A<Irak1~null><xIrak1> in BMS
    *** source: 11259596-Fig-4
  *** Tab2[Ab] infraction(membrane)[WB] is decreased irt IL1b (5 min)
    *** cells: HEK293 in BMS
    *** source: 11259596-Fig-4
  *** Tab2[Ab] infraction(membrane)[WB] is increased irt IL1b (5 min)
    *** cells: HEK293I1A<Irak1~null><xIrak1> in BMS
    *** source: 11259596-Fig-4

Subcellular fractionation. Cells at 􏰈70% confluency were either left untreated or treated with IL-1 (5 ng/ml) for the indicated times and were resuspended in 10 packed-cell volumes of ice-cold hypotonic buffer containing 10 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, and 0.5 mM DTT and homogenized on ice with 30 strokes of a Dounce homogenizer. Unlysed cells, nuclei, and cell debris were pelleted by centrifugation at 1,000 􏰄 g for 5 min. Soluble (supernatant [S100]) and particulate (pellet [P100]) fractions were generated by centrifugation at 100,000 􏰄 g for 1 h. Samples were separated by SDS-PAGE and immunoblotted with various antibodies as described above

11518704

  *** Irak1[Ab] copptby[WB] Traf6[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 4++ 10+ min
    *** comment: coppt Irak1 is upshifted
    *** source: 11518704-Fig-4a
  *** Irak1[Ab] copptby[WB] Traf6[Ab] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic fraction"
    *** times: 0+ 4+ 10+ min
    *** source: 11518704-Fig-4b
  *** Irak1[Ab] upshift[WBMS] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 3+ 10++ 30+++ 60+++ min
    *** source: 11518704-Fig-1a
  *** Irak1[Ab] upshift[WBMS] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic-fraction"
    *** times: 0 3 10 30 60 min
    *** source: 11518704-Fig-1a
  *** Irak1[Ab]IP upshift[WBMS] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 4+ 10++ min
    *** source: 11518704-Fig-4a
  *** Tab1[Ab] copptby[WB] Traf6[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic fraction"
    *** times: 0 4+ 10++ min
    *** source: 11518704-Fig-4b
  *** Tab1[Ab] infraction(cytoplasmic)[WB] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0+ 3+ 10+ 30+ 60+ min
    *** comment: cytoplasmic Tab1 mobility shift decreases with time
    *** source: 11518704-Fig-1a
  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0 3+ 10++ 30++ 60+ min
    *** source: 11518704-Fig-1a
  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0 10+ 20+ min
    *** source: 11518704-Fig-2
  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293I1A<Irak1~null><xIrak1> in BMS
    *** times: 0+ 10++ 20++ min
    *** bkg reqs: xIrak1 [omission]
    *** unaffected by: xIrak1(K293A)"KD" [substitution]
    *** inhibited by: xIrak1(104-712)"dDD" [substitution]
    *** partially inhibited by: xIrak1(del(104-198))"dUD" [substitution]
    *** bkg inhibited by: xIrak1(1-522)"dC1C2" [substitution]
    *** source: 11518704-Fig-2,3
  *** Tab2[Ab] infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0 10++ 30++ 60+ 120++ 240+ min
    *** source: 11518704-Fig-5b
  *** Tab2[Ab]IP infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0 5 10+ min
    *** source: 12242293-Fig-6
  *** Tab2[Ab]IP infraction(membrane)[WB] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0+ 5+ 10+ min
    *** source: 12242293-Fig-6
  *** Tak1[Ab] copptby[WB] Tab2[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic-fraction"
    *** times: 0 5+ 10+ min
    *** source: 12242293-Fig-6
  *** Tak1[Ab] copptby[WB] Tab2[Ab] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0+ 5+ 10+ min
    *** source: 12242293-Fig-6
  *** Tak1[Ab] copptby[WB] Traf6[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic fraction"
    *** times: 0 4+ 10++ min
    *** source: 11518704-Fig-4b
  *** Tak1[Ab] infraction(cytoplasmic)[WB] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0+ 3+ 10+ 30+ 60+ min
    *** comment: Tak1 is not detectable in the membrane fraction
    *** source: 11518704-Fig-1a
  *** Tab2[Ab] copptby[WB] Traf6[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cytoplasmic-fraction"
    *** times: 0 4+ 10++ min
    *** source: 11518704-Fig-4b

12242293


  *** Irak1[Ab] copptby[WB] Tab2[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 5++ 10+ min
    *** source: 12242293-Fig-6
  *** Irak1[Ab] copptby[WB] Tab2[Ab] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS "cyto-fraction"
    *** times: 0 5 10 min
    *** source: 12242293-Fig-6
  *** Tak1[Ab]IP infraction(cytoplasmic)[WB] is increased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0+ 2+ 5++ 10++ 30++ 60++ min
    *** comment: Tak1 phos[WBMS] is increased at 5-60 min
    *** source: 12242293-Fig-7
  *** Tak1[Ab]IP infraction(membrane)[WB] is decreased irt IL1b (times)
    *** cells: HEK293 in BMS
    *** times: 0+ 2 5 10 30 60 min
    *** source: 12242293-Fig-7
  *** Tak1[Ab]IP IVKA(Mkk6)[32P-ATP] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cyto-fraction"
    *** times: 0 2+ 5++ 10++ 30++ 60+ min
    *** source: 12242293-Fig-7
  *** Tak1[Ab]IP IVKA(Mkk6)[32P-ATP] is unchanged irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 2 5 10 30 60 min
    *** source: 12242293-Fig-7
  *** Traf6[Ab] copptby[WB] Irak1[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 4++ 10+ min
    *** source: 11518704-Fig-4a
  *** Traf6[Ab] copptby[WB] Tab2[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "membrane-fraction"
    *** times: 0 5++ 10+ min
    *** source: 12242293-Fig-6
  *** Traf6[Ab] copptby[WB] Tab2[Ab] is increased irt IL1b (times)
    *** cells: HEK293 in BMS "cyto-fraction"
    *** times: 0 5 10+ min
    *** source: 12242293-Fig-6

The subcellular fractionation method described previously (21) was used with modifications. Cells were lysed in a hypotonic buffer (10 mM HEPES [pH 7.4], 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenyl- methylsulfonyl fluoride, 0.5 mM dithiothreitol) and homogenized on ice with a Dounce homogenizer. To ensure complete breakdown of the cell membrane sheets to small membrane vesicles, 107 cells were lysed in 1 ml of hypotonic buffer (three times more buffer was used than that in the previous protocol [21]) and homogenized with 45 strokes (30 strokes used in the previous protocol [21]) Unlysed cells, cell debris, and nuclei were removed by centrifugation at 1,000 x g for 5 min. Soluble (supernatant [S-100]) and particulate (pellet [P-100]) fractions were generated by centrifugation at 100,000 􏰄 g for 1 h at 4°C. P-100 was solubilized in the Triton-containing lysis buffer and were centrifuged at 10,000 􏰄 g for 10 min. 21. Qian, Y., M. Commane, J. Ninomiya-Tsuji, K. Matsumoto, and X. Li. 2001. IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1- induced activation of NF-􏰁B. J. Biol. Chem. 276:41661–41667.



  *** xIrak1[Ab]IP IVKA(auto)[32P-ATP] is increased irt IL1b (times)
    *** cells: EL4 in BMS "nuclear-extracts"
    *** times: 0 5 90+ 120+ min
    *** inhibited by: Menadione [addition]
    *** source: 15896328-Fig-3c
  *** xIrak1[Ab]IP IVKA(auto)[32P-ATP] is increased irt IL1b (times)
    *** cells: EL4 in BMS "nuclear-extracts"
    *** times: 0 5 90+ 120++ min
    *** inhibited by: xIrak1(K239S)"KD" [substitution]
    *** source: 15896328-Fig-2b
  *** xIrak1[Ab]IP IVKA(auto)[32P-ATP] is increased irt IL1b (times)
    *** cells: EL4 in BMS "post-nuclear-extracts"
    *** times: 0 5+ 90++ 120+++ min
    *** partially inhibited by: xIrak1(K239S)"KD" [substitution]
    *** source: 15896328-Fig-2b

