Orb164257

Cell samples were processed for western blotting analysis, as previously described. The poly(vinylidene fluoride) membranes (Millipore Corporation) were incubated with the following primary antibodies at 4°C overnight: anti‐EPOR (rabbit, orb164257, Biorbyt, 1:1000), anti‐βCR (mouse, 393,281, Santa, 1:1000), and anti‐H3K9me3 (mouse, 5327, CST, 1:100). Antigen–antibody complexes were observed by enhanced chemiluminescence using an Immobilon Western blotting kit. The intensity of the bands was detected using a FluorChem®HD2 Gel Imaging System (Protein Simple). The gray values of the bands were analyzed using AlphaEase FC software (Alpha Innotech).

The cells were fixed in 4% PFA for 15 min. After washing three times in PBS, the cells were incubated with 3% BSA for 1 h. Cells were immunostained with primary antibodies against EPOR (rabbit, orb164257; Biorbyt, 1:100), anti‐βCR (mouse, 393,281; Santa, 1:100), anti‐H3K9 (mouse, 5327; CST, 1: 100), and anti‐Syne‐1 (rabbit, ab192234; Abcam, 1:100). After incubation with secondary antibodies conjugated with DyLight 488 or Cy3 (Jackson ImmunoResearch), the cells were counterstained with 4′,6‐diamidino‐2‐phenylindole. The images were digitized using an Olympus Fluoview FV1000 microscope (Olympus).