Anti klf4

The cells were rinsed briefly with phosphate-buffered saline (PBS) and fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature. The cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS, and blocked for 45–60 min with 4% bovine serum albumin in PBS at room temperature. Cells were incubated overnight at 4 °C with one of the following antibodies: anti-Oct4 (1:500; Abcam, Cambridge, MA, USA), anti-Sox2 (1:500; NB110-37235, Novus Biologicals, Littleton, CO, USA), anti-Nanog (1:500; Abcam, Cambridge, MA, USA), anti-c-Myc (1:250; bs-4963R, Bioss, Woburn, MA, USA), anti-Klf4 (1:250, bs-1064R, Bioss, Woburn, MA, USA). This was followed by incubation with the following secondary antibody: Alexa Fluor 488-labeled anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained using DAPI (1 mg/mL PBS; Invitrogen, Carlsbad, CA, USA).

The mouse xenograft tumor tissues were homogenized in the RIPA lysis buffer with PMSF, phosphatase and protease inhibitors (Keygen, Nanjing, China). The concentrations of total proteins in each sample were detected by bicinchoninic acid (BCA). Protein samples of each loading was separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) gels and transferred onto polyvinylidene difluoride membrane (Millipore, Danvers, MA, USA). The polyvinylidene difluoride membranes were blotted with anti-KLF4, anti-BMI1, anti-MMP-2, anti-MMP-9, and anti-β-actin (Bioss, London, UK). The immunoblots on the membranes were developed with the enhanced chemiluminescence reagent. The band intensity was determined by densitometric analysis using IMAGEJ software for relative quantification of protein levels by normalized with β-actin in the tumor.