Ecl primer western blotting detection reagent

The iPSCs and erythrocytes at TM day 8 were lysed with RIPA buffer (Sigma, St. Louis, MO, #R0278) in the presence of protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA, #5872S). The protein samples were treated and run with NuPAGE Bis‐Tris Mini Gel System (Life Technologies) following manufacturer's instruction. The Western blot was performed with Invitrogen iBlot Dry Blotting System with 7 minutes running time in P3 program. To detect HBB protein production, expression, we used a mouse monoclonal IgG1 (Santa Cruz Biotechnology, Dallas, TX, #sc‐21757) as a primary antibody, which specifically recognize HBB but not HBG proteins as previously reported 18. As a loading control, we also probed the blot after using anti‐human GAPDH antibodies (Cell Signaling Technology, #5174S, rabbit IgG). For secondary antibodies used for Western blot detection, we used peroxidase labeled anti‐mouse IgG(H + L) (Vector Laboratories, Burlingame, CA, #PI‐2000) or anti‐rabbit IgG(H + L) (Jackson ImmunoResearch Laboratories, West Grove, PA, #711‐035‐152), respectively. Positive signals were developed by ECL Primer Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, #RPN2232). The protein expression levels were quantified using Photoshop software based on band area and gray scale.