Sonic hedgehog

Proteins were extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific), according to manufacturer’s instructions. Western blots were then processed following standard protocols. Western blot for membrane proteins was processed using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific). We used a rabbit anti-C-terminal Shh antibody (ab53281, Abcam, 1:1 000). As a secondary antibody, we used a donkey anti-rabbit HRP-conjugated antibody (ab16284, Abcam, 1:1 000). For immunoblotting of supernatants, Shh-sorted A549 cells were cultured for 3 days and the supernatants from Shh- and Shh+ cells were concentrated using Centrifugal Filter Units (Millipore) at 14,000g for 10 minutes at 4°C. A Bradford assay was used to quantify concentrated supernatants for Western blot analysis. Sonic hedgehog (Abcam, 1:1,000) and a loading control for secreted proteins, MMP2 (OneWorldLab, 1:1,000) antibodies were used to probe blots.

Western blot analysis of mouse tissues and neurospheres was performed as previously described (Brockmann et al., 2013; Chesler et al., 2006 ). For IHC of mouse tissues, samples were fixed in 4% paraformaldehyde in phosphate buffered saline for at least 24 hr, decalcified with 0.3 M EDTA, and processed using a Leica ASP300S tissue processor. Sections were cut at 4 μM for hematoxylin and eosin staining (H&E) staining and immunohistochemistry as previously described (Chesler et al., 2006 (link)). Antibodies used were MYCN (OP-13, Merck-Millipore), Ki-67 (556003, BD Biosciences), GFAP (Z0334, DAKO), Cleaved Caspase 3 (9664, Cell Signaling Technology), Synaptophysin (180130, Life Technologies), Phospo-S10-Histone H3 (9706, Cell Signaling), phospho-AurkABC (2914, Cell Signaling), AurkA (4718, Cell Signaling), Sonic Hedgehog (ab73958, Abcam), Gli-1 (2534, Cell Signaling), and GAPDH (2118, Cell Signaling).

Protein was extracted from liver tissue or cell cultures as described [7 (link)]. Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). The buffer contains 1% proteinase and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). The nuclear and cytosolic fractions were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). The Foxo1, p-JNK, JNK, p-Akt, p-β-catenin, β-catenin, Snail, HMGB1, RIPK3, p-MLKL, NLRP3, cleaved caspase-1, Lamin B2, β-actin (Cell Signaling Technology), Gli1 (Santa Cruz Biotechnology), Sonic Hedgehog (Shh), SMO, NEK7, and TCF4 (Abcam) mAbs were used. The membranes were incubated with Abs, and then added Western ECL substrate mixture (Bio-Rad) for imaging with the iBright FL1000 (ThermoFisher Scientific). Relative quantities of protein were determined by comparing to the β-actin expression, using iBright image analysis software (ThermoFisher Scientific).