Irdye 680rd donkey anti rabbit 926 68073

Cells were collected by trypsinization, washed in 1xPBS and pellets were frozen on dry ice/EtOH bath and stored at −80C. Cells were lysed in mRIPA buffer with Protease and Phosphatase inhibitor cocktail. 20ug of protein was run on a 4–20% Mini-PORTEAN TGX precast gels in Tris-Glycine-SDS running buffer. After transfer onto the nitrocellulose membrane, and blocking in Li-Cor Intercept Blocking Buffer (TBS), blots were probed with either CTCF (Abcam 128873, 1:1000) or Rad21 (Abcam 154769, 1:1000) antibodies as well as beta-Actin (Cell Signaling 3700) in blocking buffer at 4C overnight. Membranes were then washed 3× 5min in TBST at room temperature, incubated with secondary antibodies (Li-Cor: IRDye 680RD Donkey anti-Rabbit 926–68073 and IRDye 800CW Donkey anti-Mouse, 1:10 000 diluted in blocking buffer) for 1h at room temperature. After 3× 5 min washes in TBST, and 1 wash in TBS, samples were imaged on the Li-Cor scanner.
Western blot for Ring1b and EED (Figure S3E) was reproduced from ^{25 (link)}. The protocol is the same as above but with the following antibodies: V5 tag (abcam 27671, 1:800), targeting the V5-tagged EED); HA-tag (abcam 9110) (labeling the HA-tagged RING1b, 1:800).