Rabbit anti human nanog

Proteins were extracted from the cells using RIPA buffer, resolved by SDS-polyacrylamide gels, and then transferred to poly-vinylidene difluoride (PVDF) membranes. The membranes were probed with rabbit anti-human TAZ (1:1000, cat# 83669), rabbit anti-human total caspase-3 (1:1000, cat# 9662), rabbit anti-human cleaved caspase-3 (1:1000, cat# 9661), rabbit anti-human total PARP (1:1000, clone: 46D11, cat# 9532), rabbit anti-human cleaved PARP (1:1000, clone: D64E10, cat# 5625), mouse anti-human NANOG (1:1000, cat# 4893), or rabbit anti-human SOX2 (1:1000, cat# 3579) from Cell Signaling Technology, or rabbit anti-human OCT4 (1:1000, abcam, cat# ab19857), rabbit anti-human NANOG (1:1000, clone: EPR2027(2), cat# ab109250, Abcam), mouse anti-human GAPDH (1:10000, clone: 1E6D9, cat# 60004-1-Ig, Proteintech), or rabbit anti-human α-tubulin (1:10000, cat# 11224-1-AP, Proteintech) antibodies overnight at 4 °C. HRP-linked anti-rabbit IgG (1:3000, cat# 7074, Cell Signaling Technology) or anti-mouse IgG (1:3000, cat# 7076, Cell Signaling Technology) was used and the antigen-antibody reaction was visualized by an enhanced chemiluminescence assay (ECL, Thermo). Densitometry was performed using ImageJ software. GAPDH or α-tubulin run on the same blot were used as the loading controls unless otherwise indicated.

IHC staining was performed on clinical samples through the streptavidin biotin peroxidase complex method. The antigen retrieval procedure was heated in a pressure cooker with 10 mmol/L EDTA (pH 8.0) of Dako antigen retrieval solution. Then, the samples were stained with following primary antibodies, rabbit anti-human SIRT1 (Santa Cruz), rabbit anti-human Nanog (abcam) or rabbit anti-human phospho-MEK1 (Cell Signaling). Samples were subsequently developed using EnVision method with a DAKO kits (Dako REALTM EnVisionTM). Scoring for IHC staining was performed by two independent pathologists. We quantitatively scored the tissue sections according to the percentage of positive cells and the staining intensity. We assigned the following proportion scores: 0 (0–1%), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). We rate the intensity of staining on a scale of 0 to 3: 0 for negative; 1 for weak; 2 for moderate; and 3 for strong. The positivity and intensity scores were combined to protein expression score (overall score range, 0–12). Scores were compared with overall survival, defined as the time from date of surgery to death or last known date of follow-up.