Supersignal west pico plus chemiluminescent reagent

Cells were lysed with 1× RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and stored in aliquots at − 20 °C until use. Twenty micrograms of cell lysates were mixed with an equal volume of Laemmli sample buffer, denatured by boiling, and separated by SDS-PAGE. The separated proteins were then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked using 5% BSA for 1 h at room temperature and incubated with the first antibodies against 1% BSA overnight. SOX2, OCT4, NANOG, KLF4, c-MYC, and β-actin antibodies were from Cell Signalling Technology (Beverly, MA, USA). PBX1 antibody was from Abcam (Cambridge, MA, USA) and PBX1b antibody from Santa Cruz Technology (Dallas, TX, USA). After incubation with IgG horseradish peroxidase-conjugated secondary antibodies (Cell Signalling) for 2 h at room temperature, the immunoblots were developed using SuperSignal West Pico PLUS Chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA) and imaged using ChemiDoc MP Imager (Bio-Rad).

The membrane was blocked with 5% nonfat milk in PBS for at least 1 hour at room temperature after proteins were transferred from precast 4-20% gels (BioRad). Blots were incubated for 1 hour at room temperature with 1:1000 of anti-GFP (ab290, Abcam) or 1:5000 β-actin HRP (SantaCruz). The membrane was then incubated with 1:5000 horseradish peroxidase-conjugated anti-rabbit IgG heavy and light chains (Jackson ImmunoResearch) for 1 hour at room temperature. Protein was detected using SuperSignal West Pico PLUS chemiluminescent reagent (Thermo).

Samples were mixed with SDS buffer (125 mM Tris pH 6.8; 2% SDS; 5% 2-beta mercaptoethanol; 5% glycerol; with bromophenol blue), boiled and subjected to SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes (Protan BA85, Whatman). Non-specific sites were blocked with 5% skimmed milk protein, membranes washed with PBS and then incubated with primary monoclonal antibodies against: XIRP1, (Xin-alpha (D-8); sc-166,658; 1/100; Santa Cruz Biotechnology, Insight Biotechnology Ltd., Wembly, UK); or GST, mAb 17A10 (1/100). This was followed by washing in PBS and incubation with secondary antibody (peroxidase-labelled rabbit anti-mouse immunoglobulins, P0260; 1/1000; Dako, Denmark). Alternatively, for the detection of annexin A5, pAb ANXA5 (Abcam; ab14196; 1.4 μg/mL) primary antibody followed by goat anti-rabbit Ig HRP (P0448; Dako; 1/1000) secondary antibody. All antibodies were diluted in PBS containing 0.05% Triton X, 0.1% BSA, 1% horse serum and 1% fetal bovine serum. XIRP1 and annexin A5 antibody reacting bands were detected with SuperSignal West Femto chemiluminescent reagent (Cat No: 34094; ThermoFisher Scientific) and GST antibody reacting bands were detected with SuperSignal West Pico Plus chemiluminescent reagent (Cat No: 34580; ThermoFisher Scientific) and visualized with a ChemiDoc Touch imaging system (BioRad Ltd.).