Mouse anti jnk sc 7345 mouse anti p jnk sc 6254

Unless otherwise stated, cells for western blot analysis were treated for 72 hours before being lysed. Cells were lysed using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) and protein concentration measured using the bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Inc Waltham, MA, USA). A total of 10‐20 μg of protein was separated by 4‐20% Mini‐PROTEAN TGX precast gels (Bio‐Rad, Hercules, CA, USA) and transferred to Immobilon™‐FL PVDF transfer membranes (Thermo Fisher Scientific, Inc). Membranes were dried overnight, wetted with methanol, and blocked with Odyssey Blocking Buffer (LI‐COR Biosciences, Lincoln, NE, USA) for 1 hour prior to incubation with primary antibodies, mouse anti‐RUNX1 (sc‐365644), mouse anti‐p‐RUNX1 (sc‐293146), mouse anti‐JNK (sc‐7345) mouse anti‐p‐JNK (sc‐6254) (Santa Cruz Biotechnology) and rabbit anti‐β‐actin antibody (4970S, Cell Signaling Technology) for 4 hours at room temperature (RT). Membranes were subsequently washed with TBST and incubated with secondary antibodies (IRDye 680RD donkey anti‐rabbit and IRDye 800CW donkey anti‐mouse (dilution 1:4000) (LI‐COR Biosciences). After rinsing twice with TBST, immunoreactive bands were visualized using the Odyssey Infrared Imaging System, and band intensities normalized to β‐actin were quantified using Image Studio version 2.1 (LI‐COR Biosciences).