Rabbit anti oct4

Cells were lysed in Tris-HCl pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, Na pyrophosphate 2 mM, and protease inhibitors. Lysates were separated on 10% or 12% acrylamide gel and immunoblotted using standard procedures. Rabbit anti-OCT4, #2750 (Cell Signaling Technology Inc., Danvers, MA), rabbit anti-Nanog, PA1-097 (ThermoFisher Scientific, Rockford, IL), mouse anti-β-3-Tubulin (TU-20), #4466 (Cell Signaling Technology Inc.), mouse anti-GFAP, MAB360 (Merck Millipore, Darmstadt), rabbit anti-HspA1A, sc-33575 (Santa Cruz Biotechnology, CA), rabbit anti-PCNA, #13110 (Cell Signaling Technology Inc.), rabbit anti-NPM, #3542 (Cell Signaling Technology Inc.), mouse anti-GAPDH, ab8245 (AbCam, Cambridge, UK), rabbit anti-Hsp60, #D307 (Cell Signaling Technology Inc.), and HRP-conjugated secondary antisera (Santa Cruz Biotechnology, CA) were used followed by enhanced chemiluminescence (ECL Amersham, Amersham, UK) and images were acquired using BioRad ChemiDoc MP Imaging System (BioRad, Hercules, CA). Densitometric analysis was performed using the BioRad associated Image Lab Software (BioRad, Hercules, CA). Values are expressed as fold over internal control, represented by GAPDH or Hsp60, in the case of NPM, whose expressions were not significantly modulated in the proteome profiles.

Cells were washed with ice-cold PBS and lysed in RIPA lysis buffer with freshly added cocktail protease inhibitor (Thermo Scientific, Rockford, IL, USA) on ice. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, County Cork, Ireland). The membranes were blocked with 5% fat-free milk in TBST at room temperature for 60 min and then incubated with indicated primary antibodies followed by incubation with peroxidase-conjugated secondary antibodies (Thermo Scientific, Rockford, IL, USA) at room temperature for 60 min. The protein bands were detected and analyzed with an enhanced chemiluminescence kit (Amersham, Marlborough, MA, UK) using Bio-Rad ChemiDoc XRS⁺ Imaging System according to the manufacturer’s instructions. The Primary antibodies were used as follows: mouse anti-β-Actin (Proteintech, Wuhan, China), rabbit anti-ERβ (Bioworld Technology, Louis Park, MN, USA), rabbit anti-OCT4 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-NANOG (Cell Signaling Technology, Danvers, MA, USA).

For detection of stage-specific markers, cells were grown and differentiated in 96 well plates (μClear, Greiner). Cells were rinsed twice with PBS, fixed in 4% paraformaldehyde (USB) for 15 min at room temperature (RT) and then washed twice with PBS and blocked for 30 min at RT in 1% BSA (Life Technologies) and 0.1 mg/ml human IgG (Sigma) in perm/wash buffer (BD). Cells were subsequently stained for 2 hours at RT or overnight at 4°C with rabbit anti-OCT4 (Cell Signalling), mouse anti-SOX17 (Abcam) or isotype control antibodies diluted in perm/wash buffer. Cells were washed with perm/wash buffer and incubated at 4°C in the dark with goat anti-mouse-FITC and chicken anti-rabbit-Cy5 (Molecular probes) diluted 1:400 in perm/wash buffer. After 1h of incubation, cells were washed with PBS and incubated with Hoechst 33342 (Life Technologies) for 15 min at room temperature. After final washing with PBS 96 well plates were then imaged with IN Cell Analyzer 2000 (GE Healthcare).

Western blot analysis was performed as previously described.^{52 (link)} Blots were probed with 1/1000 rabbit anti-Oct4 (#2788), 1/1000 rabbit anti-MAP2 (#4542) (Cell Signaling Technology, Beverly, MA, USA), 1/500 rabbit anti-Foxj3 (#SAB2100844), 1/500 rabbit anti-Zbtb18 (#SAB2103436), 1/1000 mouse anti-Nestin (#MAB5326), 1/1000 rabbit anti-Brachyury (#B8436), 1/2000 mouse anti-β-actin (#A5441) (Sigma-Aldrich), 1/1000 goat anti-Flk1 (VEGF receptor 2, #AF644; R&D Systems, Minneapolis, MN, USA), 1/1000 mouse anti-Sox17 (#ab192453), 1/1000 rabbit anti-Foxa2 (#ab108422) (Abcam, Cambridge, MA, USA), and 1/1000 mouse anti-Tuj1 (neuronal class III β-tubulin, #MMS-435P; Covance, Princeton, NJ, USA). Immunoblots were revealed by autograph using SuperSignal west pico substrate (Thermo Fisher Scientific). The relative intensity of the protein bands was quantified using Image J software (NIH, Bethesda, MD, USA) and calculated by samples normalized to the controls. All data were presented as mean±S.D. and derived from three independent experiments.

Cells were seeded in 24‐well plates over coverslips (1 × 10³ cells/well) and treated as described in Figure legend. Then, cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X‐100 in PBS, blocked with 3% BSA/PBS and stained with primary antibodies: rabbit anti‐Ki67 (Abcam), mouse anti‐NF‐κB (Santa Cruz Biotechnology), mouse anti‐β‐catenin (Santa Cruz Biotechnology), mouse anti‐NANOG, rabbit anti‐Oct‐4 and mouse anti‐SOX‐2 (Cell Signaling Technology, Danvers, MA, USA). Samples incubated in 3% BSA/PBS only represented negative controls (Supplementary Figure S3). The corresponding FITC‐coupled secondary antibodies (Sigma‐Aldrich) and 1 ng/mL of nuclear dye DAPI (Sigma‐Aldrich) were added during 2 hours. Samples were examined using an epi‐fluorescent microscope (Olympus, Japan).