Anti shh

The Western blot analysis was performed as we previously described [64 (link)]. Briefly, cells were washed with phosphate buffer saline (PBS), lysed in RIPA Buffer and centrifuged. Equal amounts of protein determined by the BCA method (Pierce BCA protein assay kit, Waltham, MA, USA) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Burlington, MA, USA). Membranes were incubated overnight at 4 °C with specific antibodies followed by incubation with HRP-conjugated anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) secondary antibody (1:10,000 dilution; Cell Signaling Technology, Inc.) for 30 min at room temperature. Signals were established using a chemiluminescence detection kit (Pierce ECL Western blotting substrate, Thermo Scientific, Rockford, IL, USA). Band intensities were quantitated using the program ImageJ (NIH) [65 (link)]. The primary antibodies used were as follows: Anti-vimentin (1:750; cat. no. sc-6260), anti-E-cadherin (1:500; cat. no. sc-597780), anti-N-cadherin (1:500; cat. no. sc-393933), anti-SHH (1:1000; Cell Signaling, cat. no. 2707), anti-GLI1 (1:000; Cell Signaling, cat. no. 3538), anti-β-actin (1:750; cat. no. sc-47778) and anti-GAPDH (1:750; cat. no. sc-32233).

General laboratory chemicals were obtained from Sigma-Aldrich or Merck Millipore (Darmstadt, Germany). TNF-α and TRAIL were purchased from PeproTech Inc (Rocky Hill, NJ, USA). Pam3CSK4, LPS and R848 were purchased from ImmunoTools (Friesoythe, Germany). Anti-β-ACTIN antibody was bought from Sigma-Aldrich. Anti-p53 and anti-COP1 antibodies were obtained from Santa Cruz Biotechnology Ltd (Dallas, Texas, USA). Anti-SHH, anti-GLI1, anti-PTCH1, anti-NUMB, anti-Ser9 phospho GSK-3β and anti-UbK48 were purchased from Cell Signaling Technology (Danvers, MA, USA). Annexin V- fluorescein isothiocyanate (FITC) was from Miltenyi Biotech (Bergisch Gladbach, Germany). HRP conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA).

Proteins (60 μg) were separated using 8% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), and incubated with primary antibodies overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, KangChen, Shanghai, China) and the signals were visualized using an enhanced luminescence (ECL) Kit (Thermo Fisher Scientific). The following commercial antibodies were used: anti-SHH (Cell Signaling Technology, Danvers, MA, USA), anti-GLI1 (Cell Signaling Technology), anti-SOX2(Cell Signaling Technology), anti-NANOG (Cell Signaling Technology), anti-MYC antibody (Abcam, Cambridge, UK) and anti-β-actin (Cell Signaling Technology). All antibodies were used at a dilution of 1:1000.