Co-immunoprecipitation (co-IP) and reverse co-IP assays were performed using Dynabeads Protein G (Thermo Fisher Scientific). K562 and HEK293 cells that stably overexpressed chorein8 (link) were lysed with Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). K562 cells that were subcultured at 1 × 106 cells/mL and incubated for 24 hours were used. The cell lysates (input) were used for the Dynabeads-antibody complex and Dynabeads-IgG complex. The cell lysate was diluted 5 times with 1× Tris-buffered saline because delicate surfactant conditions were required to maintain the IP interaction. The cell lysate and each bead were incubated for 2 hours at room temperature.
Protein samples were analyzed by immunoblotting using rabbit anti-chorein (HPA021662; Atlas Antibodies, Bromma, Sweden) and rabbit anti-XK protein (HPA019036; Atlas Antibodies) primary antibodies, which show no cross-reactivity with spectrin. Donkey anti-rabbit IgG, HRP-linked whole Ab (GE Health care, Little Chalfont, England) and VeriBlot for IP Detection Reagent (HRP) (ab131366; Abcam, Cambridge, UK) were used as secondary antibodies. Proteins were visualized using ECL Prime Western Blotting Detection Reagent (GE Health care), and images were recorded with a digital analyzer (FUSION-SOLO.7S.WL; Vilber Lourmat, Marne-la-Vallée, France).
Protein samples were analyzed by immunoblotting using rabbit anti-chorein (HPA021662; Atlas Antibodies, Bromma, Sweden) and rabbit anti-XK protein (HPA019036; Atlas Antibodies) primary antibodies, which show no cross-reactivity with spectrin. Donkey anti-rabbit IgG, HRP-linked whole Ab (GE Health care, Little Chalfont, England) and VeriBlot for IP Detection Reagent (HRP) (ab131366; Abcam, Cambridge, UK) were used as secondary antibodies. Proteins were visualized using ECL Prime Western Blotting Detection Reagent (GE Health care), and images were recorded with a digital analyzer (FUSION-SOLO.7S.WL; Vilber Lourmat, Marne-la-Vallée, France).