Iodixanol gradients were performed similarly to previously described (27) . Briefly, HEK293 cells expressing H2a-RFP (untreated or treated with Bz) were washed with PBS, resuspended in a homogenization buffer (0.25M Sucrose, and 10mM HEPES pH7.4). The cells were passed through a 21G needle 5 times before homogenization in a Dounce homogenizer (low clearance pestle, 30 strokes). The homogenates were centrifuged at 1000xg for 10 minutes at 4˚C to remove nuclei and cell debris and the supernatants were loaded on top of an iodixanol gradient (10 to 34%). The gradients were ultra-centrifuged at 24,000 rpm (98,500g, Beckman SW41 rotor) at 4˚C for 16 hours.
1ml gradient fractions were collected from top to bottom, precipitated with 10% TCA and run on SDS-PAGE.
1ml gradient fractions were collected from top to bottom, precipitated with 10% TCA and run on SDS-PAGE.
