Anti β tubulin

Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously (McClintock et al., 2006 (link)). The membranes were incubated with primary antibodies: anti-lamin A/C [kindly provided by Dr. N. Chaudhary (1/5000) (Chaudhary & Courvalin, 1993 (link)), anti-progerin antibody (clone S9, 0.1 μg mL⁻¹) (McClintock et al., 2007 (link)), anti-prelamin A antibodies (sc-6214, Santa Cruz Biotechnology, 1/1000), anti-proteasome S20 subunit C2 (ab22665, Abcam, 1/000), anti-Hsp27 (ab2790, Abcam, 1/2000), anti-ubiquitin (sc-8017, Santa Cruz Biotechnology, 1/3000), anti-LC3B (Sigma-Aldrich, 1/4000), anti-53BP1 (A300-272A, Bethyl, 1/1000)], anti-FHL-1 (sc-133580, Santa Cruz Biotechnology, 1/1000) anti-Rad51 (NBP2-32622, Novus Biological, 1/1000) anti-β-actin (Sigma-Aldrich, 1/5000) and anti-β-tubulin (Thermo Fisher, 1/2000). Then washed and incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized using a chemiluminescence detection system (ECL substrate; BioRad). Signals were analyzed with image lab software (BioRad). Protein signals were quantified by normalizing to β-actin as indicated.

Human recombinant ARG-1 was kindly provided by Hepius Biotech AG. Prior to use, ARG was activated with 0.5 mM MnCl₂ at 50 °C for 10 min. 1 U/mL of ARG was calculated equivalent to 0.001 mg/mL. Recombinant human insulin was purchased from Sigma-Aldrich. Antibodies were purchased from commercial sources as follows; anti-Insulin receptor (InsR, ab131238, Abcam), anti-ARG-1 (#93,668, Cell Signalling Technology), anti-β-Tubulin (#322,600, ThermoFisher), anti-mouse IgG-horseradish peroxidase (HRP) (#7076, Cell Signalling Technology) and anti-rabbit IgG-HRP (#7074, Cell Signalling Technology).

P. vitticeps embryos at oviposition were fixed overnight in 4% paraformaldehyde (PFA) in PBS at 4°C, dehydrated though a series of methanol/PBS solutions (25, 50, 75, and 100% methanol), and stored at −20°C until hybridization or immunofluorescence. Whole mount in situ hybridization (WMISH) was performed according to our previously published protocol (Di-Poï et al., 2010 (link)) at a temperature of 68°C. New species-specific digoxigenin-labeled antisense riboprobes corresponding to P. vitticeps Dlx2 (831 bp, 3′ UTR region) and Sox10 (837 bp, 3′ UTR region) genes were designed based on publicly available P. vitticeps genome sequence (Georges et al., 2015 (link)). Corresponding sense riboprobes were used as negative controls. For immunofluorescence, embryos were embedded in paraffin following alcohol dehydration and then sectioned at 7 μm. Staining was performed as previously described (Di-Poï and Milinkovitch, 2013 (link)) using heat-induced epitope retrieval, primary antibodies known to recognize reptile and/or chicken epitopes (anti-β-tubulin: 1:400, Thermo Fischer Scientific; anti-ISLET-1: 1:700, Abcam), and Alexa Fluor-conjugated secondary antibodies (Alexa Fluor-488 or−568, Life Technologies). Samples were mounted with Fluoroshield mounting medium (Sigma) containing 4′,6′-diamidino-2-phenylindole (DAPI).

For preparation of zebrafish protein samples, 5 dpf embryos were homogenized in cold PBS with protease inhibitors (Roche) using syringe (1 ml) and needle (size 23G). The deyolked body fragments were collected and heated in whole cell lysis buffer (20 mM NaF, 1 mM DTT, 1 mM EDTA, 0.1 mM Na3VO3, 10% glycerol, 0.5% Nonidet P40, 280 mM KCl, 20 mM Hepes pH7.9) at 100°C for 10 minutes. Lysate supernatant was used for western blot analysis according to the standard protocol [48 (link)]. In this study, primary antibodies, anti-Taz (CST, 1:1000) and anti-β-Tubulin (Thermo, 1:1000) were used, while anti-mouse-IgG-HRP (Thermo, 1:5000) and anti-rabbit-IgG-HRP (Thermo, 1:5000) worked as secondary antibodies.

Equivalent amounts of protein from each sample type were heated for 10 minutes to 90 °C in reducing buffer and then analyzed by Tris-Glycine SDS-PAGE (BioRad; Hercules, CA) followed by semi-dry protein transfer onto PVDF membrane (BioRad; Hercules, CA). Blots were blocked with Tris Buffered Saline with 0.1% tween (TBST) and 5% non-fat dry milk overnight at 4 °C, followed by another overnight incubation at 4 °C with one of the following primary antibodies diluted into TBST: anti-Caspase-3 (ab13847, Abcam; Cambridge, MA), anti-β-Tubulin (#PA1-41331, Thermo Fisher Scientific, Waltham, MA; 1:1000). Secondary antibody was anti-rabbit IgG HRP (#sc-2357, Santa Cruz Biotechnology, Dallas, TX), and SuperSignal Western Blotting Substrates (Dura and Femto; Thermo Fisher Scientific; Waltham, MA) were used for protein detection. Chemiluminescence was detected using a GE ImageQuant LAS 4000, and ImageJ was used to quantify band intensity from the digital images. Full images of cropped western blots is included in Supplemental Fig. 3.