Protein Extraction and Immunoblot Analysis Protocol

Cellular protein extraction and immunoblot analysis were performed as previously described [71 (link),72 (link)]. Briefly, cellular proteins were extracted using a detergent containing CHAPS lysis buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane. The membrane was hybridized to primary antibodies: anti-ERCC1 (D61F5, Cell Signaling, Danvers, MA, USA), anti-OCT4 (11263-1-AP, Proteintech, Rosemont, IL, USA), anti-Nanog (EPR2027(2), Abcam, Cambridge, MA, USA), anti-SPC25 (ab20679, Abcam, Cambridge, MA, USA), and anti-GAPDH (AF7021, Affinity Biosciences, Cincinnati, OH, USA). The membrane was subsequently incubated with a secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotech, CA, USA). The membranes were developed using an ECL developing solution (Merck Millipore, MA, USA) followed by autoradiography. All immunoblot experiments were performed at least two times. The original blots are shown in the Figure S1, and one typical result is presented. The protein expression shown in each band was quantified after normalization to the GAPDH expression level. The error bars shown in the relevant figures indicated the standard deviation of the quantification results.