Anti sox9

Cells of different experimental groups were cultured on coverslips as described previously. Cells were fixed in Saint-Marie’s fixative (99% ethanol and 1% anhydrous acetic acid) for 1 h. After washing in 70% ethanol, non-specific binding sites were blocked in PBST supplemented with 1% bovine serum albumin (BSA, Amresco LLC, Solon, OH, USA) at 37 °C.
For immuncytochemical staining, cells were incubated with anti-Sox9 (Abcam, Cambridge, UK; Cat. No.: ab26414) at a dilution of 1:600, and anti-GluN1 (Cell Signaling, Danvers, MA, USA; Cat. No.: 5704) antibodies at a dilution of 1:500 at 4 °C overnight. Primary antibodies were visualised with anti-rabbit Alexa Fluor 555 or anti-mouse Alexa Fluor 488 secondary antibodies (Life Technologies Corporation, Carlsbad, CA, USA; Cat. No.: A27039; A28175) at a dilution of 1:1000. Cultures were mounted in Vectashield Hard Set mounting medium (Vector Laboratories Ltd.) containing DAPI to visualise the nuclei of cells. For investigation of subcellular localization of Sox9 and GluN1, images were taken with an Olympus FV1000S confocal microscope (Olympus Co. Tokyo, Japan) using a 60× oil immersion objective (NA: 1.3). For excitation, laser lines of 543 nm and 488 nm were used. The average pixel time was 4 μs. Images of Alexa Fluor 555, Alexa Fluor 488 and DAPI were overlaid using Adobe Photoshop version 10.0 software.

The tumor cells were lysed by using RIPA buffer with Phenyl methane sulfonyl fluoride (PMSF). The same amount of protein was separated with 10% SDS-PAGE gel and then was transferred onto PVDF membranes (Biotech). Subsequently, 5% skim milk (diluted in PBS) was used to blocked the PVDF membranes for 2 h at RT. And then the membranes were incubated with anti-Gli1 (Santa), anti-CD44 (Abcam), anti-LSD1 (ZSGB-BIO), anti-Sox9 (Abcam), anti-β-actin (Abcam). The next step is to incubate anti-rabbit/mouse for 2 h. Detection was performed by the ECL kit.

Harvested fresh samples were ground in liquid nitrogen and lysed with mammalian protein extraction reagent (Pierce, Rockford, IL, USA) supplemented with complete protease inhibitor. The Pierce BCA Protein Assay Kit (Pierce) was used to determine the total protein content, which was adjusted to 10 μg/μl. Samples (10 μl) were loaded onto 10-14% SDS polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA). The membranes were blocked using 5% skim milk in 0.05% Tris-buffered saline/Tween 20 (TBST) for 1 hour. Then, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-Sox9 (Abcam, Cambridge, MA, USA, 1 : 500) and anti-β-actin (Cell Signaling, Danvers, MA, 1 : 1000). After further washes, the chemiluminescence of the blot was processed with the ECL western blotting substrate (Pierce).