Human embryonic kidney 293 (HEK 293) cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum and transfected with myc-tagged HSP70 plasmid using the Mammalian Cell Transfection Kit. Cells were harvested and lysed in 1 ml of SDS lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 0.5% TX-100, 1 mM Na 3 VO 4 , 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM PMSF, 1% SDS) for 30 min on ice, followed by centrifugation at 14000 rpm for 15 min at 4°C to remove the insoluble fraction. The protein concentration of the supernatant was determined using the Bio-Rad DC protein assay reagent with bovine serum albumin (BSA) as standard. Equal amount of protein (750 µg) was immunoprecipitated with anti-myc and collected with agarose-coupled secondary antibody. To the agarose beads containing the immunoprecipitated HSP70, 50 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 2 mM DTT, 2 mM ATP) was added containing 100 ng E1, 200 ng E2 (UbcH7), and 100 µg of E3 (Flag-tagged TRAF6) along with 5 µg GST-WT-Ub, GST-K29R Ub, GST-K48R Ub, GST-K63R Ub, or K63 Ub. Control samples without HSP70, E1, E2, E3, or GST-WT-Ub were also included. Reactions were carried out by continuous shaking at 37°C for 2 hours and then washed three times with reaction buffer. The proteins were released by boiling for 2 min in SDS-PAGE sample buffer, separated on 7.5% SDS-PAGE and Western blotted for anti-ubiquitin.