Anti sox 2 antibody

DT and TMZ were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The proteasome inhibitor MG132 was purchased from Calbiochem (San Diego, CA, USA). Monoclonal antibodies were purchased from the following companies: anti-CA9, anti-PARP-1, and anti-uPA antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-HIF-1α and anti-HIF-1β antibodies from BD Biosciences (San Jose, CA, USA); anti-phospho-JNK and anti-JNK antibodies from Promega (Madison, WI, USA); anti-actin antibody from ICN (Costa Mesa, CA, USA); anti-CD133 and anti-VEGF antibodies from Abcam (USA); anti-β-Tubulin (Tuj1) antibody from Covance (USA); anti-Nestin antibody from Chemicon (USA); anti-SOX-2 antibody from R&D Systems (Minneapolis, MN, USA); and anti-Bmi1, anti-Musashi, anti-GFAP, anti-phospho-ERK, anti-ERK, anti-phospho-p38, anti-p38, anti-MMP-2, anti-MMP-9, and anti-phospho-Akt antibodies from Cell Signaling (Beverly, MA, USA).

MCF-7/S0.5 and fulvestrant-resistant cells were fixed in 4% formalin, paraffin-embedded, and mounted in 4-μm sections on glass slides. Antigen retrieval was performed by boiling sections in T-EG solution/TRS buffer (Dako). Sections were incubated with anti-SOX2 antibody (#AF2018, R&D Systems) for one hour at room temperature. PowerVision Poly-HRP was used as the detection system. Microscopy of cells was performed using a Leica DMLB microscope (× 100 or × 200/numerical aperture (NA) 1.25, Leica Microsystems) using LasV3.6 acquisition software.

Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1 mM Na₃VO₄, 10 mM NaF, and protease inhibitors). Protein samples were separated on an SDS polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA). Membranes were blocked using 1% BSA and incubated with the following primary antibodies: anti-Sox2 antibody (R&D Systems), anti-FLAG antibody (Sigma-Aldrich), anti-O-GlcNAc antibody RL2 clone (Santa Cruz), and anti-β-actin antibody (Sigma-Aldrich). The membranes were then incubated with anti-rabbit IgG (light chain specific, Jackson Immunoresearch, West Grove, PA) or anti-mouse IgG (Cell Signaling, Danvers, MA) conjugated with horseradish peroxidase (HRP) as a secondary antibody. The membranes were then washed and developed with ECL Plus reagents (GE Healthcare, Chicago, IL).