Secondary antibodies conjugated to hrp

Leukemia cells (HL-60, NB4, THP-1 and U937) were seeded in 24-well plates at 5 × 10⁵ cells/well in which E5 was added at different concentrations for incubation of 24 h. Collected and washed with PBS, cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) supplemented with protease inhibitors (Sigma-Aldrich, USA) on the ice for 30 min. The lysates were clarified by centrifugation at 15000 rpm for 10 min. Equal protein (30 µg) was loaded and separated on 12% polyacrylamide gels (Applygen, Beijing, China), and transferred to PVDF membranes (0.2 µm; Millipore, Bedford, MA). Cleaved caspase-3 and β-Actin were probed with specific primary antibodies (Cell Signaling Technology, Beverly, MA) and secondary antibodies conjugated to HRP (Zhongshan Goldenbridge biotechnology Co, Beijing, China). The immunocomplex on the membrane was visualized using Image Quant LAS 4000 (GE Healthcare) with Immobilon ® Western HRP Substrate luminol reagent and peroxide solution (Millipore, Billerica, MA).

Proteins were isolated and analyzed according to previously described methods (20 (link), 25 (link)). The primary antibodies used were as follows: NR4A1, Survivin (Proteintech, Wuhan, China), phospho-eIF2α (Ser-51), eIF2α, CHOP (Cell Signaling Technology, Boston, MA), Caspase3–17-kDa, Bcl-2, Lamin A (Bioworld Technology, Louis Park, MA), and growth arrest and DNA damage-inducible protein-34 (GADD34; Biosynthesis Biotechnology Co., Beijing, China). Secondary antibodies conjugated to HRP (Zhongshan Golden Bridge Biotechnology, Beijing, China) were used for Western blotting.