Horseradish peroxidase conjugated secondary antibody

The Western Blot protocol was performed as previously described (14). Briefly, 100μg of protein was loaded for H727 and H720 lysates. Oct-4, Sox-2, Nanog, CD44, ALDH1 (Cell Signaling Technology, Toronto, ON, Canada) antibodies were used at 1:1000 dilution. Secondary horseradish peroxidase conjugated antibodies (Jackson Immunoresearch, West Grove, PA, USA) were used at a dilution of 1:6000 and signal was detected with the Supersignal chemiluminescence detection system (Pierce Biotechnology, Rockford, IL, USA). GAPDH served as the loading control. Signals were quantified by densitometry relative to untreated values.

Cells were lysed in ice-cold M-PER lysis buffer purchased from Thermo Fisher Scientific. Cell lysates were clarified by centrifugation (20 min at 15,000 rpm at 4°C) and protein concentration determined using the BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of protein were separated on an SDS-PAGE gel. The gel was electrophoretically transferred to a Hybond PVDF transfer membrane (Millipore, Bedford, Massachusetts) and incubated with primary and secondary antibodies according to the Supersignal^® West Pico chemiluminescence protocol (Pierce, Rockford, IL). Antibody specific for FLAG (DYKDDDDK epitope) tag was obtained from Takara Bio, Inc. (Shiga, Japan) and antibody specific for SOX2, CDKN1A, VIM, AKT and phosphorylated AKT (Ser473) were purchased from Cell Signaling Technology (Beverly, MA). Antibody specific for TP53 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and antibody specific for MSH6 and BMP2 were purchased from Proteintech Japan (Tokyo, Japan). Secondary horseradish peroxidase-conjugated antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Each experiment was repeated at least three times and the representative data is displayed.

RIPA buffer (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0) with a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) was used to obtain the cell lysates. The Bradford assay was used to evaluate the protein concentration in each sample. Proteins (25 μg) were analyzed by SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane that was then incubated overnight with primary antibodies.
Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-PCNA (Cell Signaling) and mouse anti-β-actin (Cell Signaling) were used as primary antibodies.
We used secondary horseradish peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to visualize the protein bands by using the Clarity ECL chemiluminescence substrate (Bio-Rad) that were then quantified using ImageJ software. Each measure was normalized with respect to β-actin.

Cells were lysed with RIPA extraction buffer (MBiotech, Seoul, Korea) supplemented with a CompleteMini protease inhibitor tablet (Roche, Indianopolis, IN, USA). 100ug of protein was loaded for SH-SY5Y lysates, and 20 μg for NT2/D1 lysates. OCT4, SOX2 (Cell Signaling Technology, Toronto, ON, Canada) and Nanog (Cell Signaling Technology, Toronto, ON, Canada) antibodies were used at 1/1000 dilution. Secondary horseradish peroxidase conjugated antibodies (Jackson Immunoresearch, West Grove, PA, USA) were used at a dilution of 1/6000 and signal was detected with the Supersignal chemiluminescence detection system (Pierce Biotechnology, Rockford, Il, USA).