Sc 166860

Whole-cell lysates were prepared as previously described [37 (link)]. Lysates (50 μg) were subjected to an immunoblot analysis using SDS-PAGE (12.5%), and electrotransferred onto nitrocellulose filters. The filters were incubated with primary antibodies for the epidermal growth factor receptor (EGFR; Cell Signaling Technology, Beverly, MA, USA), phosphorylated EGFR (pTyr992) (pEGFR; Cell Signaling Technology), vascular endothelial growth factor (VEGF; SC-374628, Santa-Cruz), nucleostemin (NS; SC-166460, Santa-Cruz), focal adhesion kinase (FAK; SC-271126, Santa-Cruz), phosphorylated FAK (pTyr397) (pFAK; P00151, Boster Immunoleader, Pleasanton, CA, USA) c-SRC (25978-1-AP, Proteintech Group, Rosemont, IL, USA) phosphorylated c-SRC (pTyr530) (pc-SRC; SC-166860, Santa-Cruz), and CDX2 (Oncogene Research Products, Cambridge, MA, USA). They were then incubated with peroxidase-conjugated IgG antibodies (MBL), and visualized using an enhanced chemiluminescence Western-blot detection system (Amersham, Aylesbury, UK). An anti-tubulin antibody was used as a loading control (Oncogene Research Products, Cambridge, MA, USA).

Antibodies were generated against Myc (M4439, mouse monoclonal 1:200, Sigma-Aldrich), Ptch1 (sc-293416, mouse monoclonal 1:200, Santa Cruz), Ptch2 (PA1-46223, rabbit polyclonal 1:200, Invitrogen), Gas1 (AF2636, goat polyclonal 1:500, R&D; PA5-48298, rabbit polyclonal 1:500), Boc (AF2385, goat polyclonal 1:500, R&D; MAB20361, mouse monoclonal 1:500, R&D), Cdon (GTX105422, rabbit polyclonal 1:500, GeneTex), Smo (sc-166685, mouse monoclonal 1:200, Santa Cruz, A3274, rabbit polyclonal 1:200, ABclonal), Shh (sc-365112, mouse monoclonal 1:200, Santa Cruz), Gli3 (AF3690, goat polyclonal 1:200, R&D), WDR11 (ab175256, rabbit polyclonal 1:200, Abcam), SSEA1(MC-480, mouse monoclonal 1:200, DSHB), Stella (ab19878, rabbit polyclonal 1:200, Abcam), phospho-Src, (sc-166860, mouse monoclonal 1:500, Santa Cruz), phospho-Creb (sc-81486, mouse monoclonal 1:500, Santa Cruz), Arl13b (17711-1-AP, rabbit polyclonal 1:500, Proteintech), IFT88 (13967-1-AP, rabbit polyclonal 1:500, Proteintech), CEP164 (sc-515403, mouse monoclonal 1:200, Santa cruz), gamma‑tubulin (T6557, mouse monoclonal 1:500, Sigma), b-actin (4967L, rabbit polyclonal 1:500, CST). HRP-conjugated secondary antibodies used were goat anti-mouse IgG (sc-516102, 1:1000, Santa Cruz), goat anti-rabbit IgG (ab6721, 1:1000, Abcam), donkey anti-goat IgG (ab6741, 1:1000, Abcam).