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Uncharged anti-FLAG M2 affinity column (lane 1), or anti-FLAG M2 affinity columns charged with FLAG–Smt3 SUMO-activating enzyme (lane 2), FLAG–DmUbc9 SUMO-conjugating enzyme (lane 3) or FLAG–Hsp82, were loaded with Rpn10/p54. The columns were eluted with an excess of FLAG peptide and analysed by immunoblotting with mAb 170.
