Anti β galactosidase

Total protein lysates were obtained by homogenizing the cells in lysis buffer (10 mM Tris/HCl, pH 7.6, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, mPIC protease inhibitor cocktail 1:500). Samples were incubated for 30 min on ice and centrifuged at 10,000 g for 5 min at 4°C. The protein concentration was determined by BCA assay. Prior to separation by SDS-PAGE, 50 isolated ZPs, cells, or tissue lysates were mixed with 4 × SDS loading buffer [200 mM Tris/HCl, pH 6.8, 8% SDS (w/v), 4% β-mercaptoethanol (vol/vol), 50% glycerol, 0.04% bromophenol blue] and heated for 5 min at 95°C. For Western blot analysis, proteins were transferred onto PVDF membranes (Merck Millipore, Billerica, United States), probed with antibodies, and analyzed using a chemiluminescence detection system. For deglycosylation, 50 ZPs were incubated for 1 h with PNGase-F (New England Biolabs) according to the manufacturer’s instructions.
Primary antibodies: anti-β-galactosidase (1:1000; Molecular Probes), anti-α-tubulin (1:5000; Sigma-Aldrich), antibodies against mouse ZP glycoproteins were isolated from the supernatant of hybridoma cell lines (mZP1: ATCC CRL-2464, mZP2: ATCC CRL-2463, mZP3: ATCC CRL-2463) and diluted 1:1000. Secondary antibody: goat-anti-rat, HRP conjugated (1:5000, Dianova), donkey-anti-rabbit, HRP conjugated (1:5000, Dianova).

The immunohistochemical procedure initiated with a 30 min dehydratation at 37°C followed by a 30 min hydration in 0.1 PBS and 1 h blocking in a 0.3% triton in 0.1 PBS with 10% normal goat serum both at room temperature (RT). The following primary antibodies diluted in a blocking solution with 0.1% triton were used: mouse monoclonal anti-ataxin-3 (1H9, Chemicon, Temecula, CA, USA; 1∶5000; 48 h, 4°C), rabbit polyclonal anti-β-galactosidase (Molecular Probes, Invitrogen; 1∶1000; 48 h, 4°C), rabbit polyclonal anti-DARPP-32 (Chemicon,Temecula, CA, USA; 1∶5000, 48 h, 4°C), rabbit polyclonal anti-calbindin D-28K (Chemicon,Temecula, CA, USA; 1∶1000, 48 h, 4°C). Sections were then incubated in secondary antibody, goat-anti rabbit and/or mouse conjugated to alexa 488 or 594 (Invitrogen) for 2 h/RT e and then mounted in mowiol (Sigma) with 4′,6′-diamidino-2-phenylindole.Fluorescence images were acquired with a Zeiss Axiovert 200 imaging microscope or LSM Zeiss microscope for double staining experiments.

NIH3T3 cells were cultured in DMEM containing 10% Bovine Calf Serum (ATCC). HEK 393T and Sufu -/- MEF cells were cultured in DMEM with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich). Shh treatment was done by serum starvation for 24 hours (0.5% Bovine Calf Serum), then adding a recombinant mouse Shh N-terminal fragment (R&D Systems #464-SH) at 1 ug/ml overnight. Smoothened agonist SAG (Sigma Aldrich) treatment was done at 200 ng/ml for 8–12 hours. Cell transfections were performed using PolyJet in vitro DNA Transfection Reagent (SignaGen) following manufacturer’s instruction. Immunoprecipitation, immunostaining, and western blot analyses were carried out as described previously [47 (link)]. The antibodies used in this study are listed as follows: anti-Myc (9E10, Santa Cruz Biotechnology), anti-β-galactosidase (A11132, Life Technologies), anti-acetylated tubulin (T7451, Sigma Aldrich), anti-mGli2 (AF3635, R&D Systems), anti-mGli3 (AF3690, R&D Systems), anti-mKapβ2 (Ab10303, Abcam), anti-α-tubulin (T9026, Sigma Aldrich), anti-Histone3 (Ab1791, Abcam), and anti-BrdU (B8434, Sigma Aldrich).