Fluorescently conjugated secondary antibodies

Embryos used for the analysis of Wnt protein expression were cultured and microinjected using the methods described above. Five YFP-positive embryos were flash-frozen at the mid gastrula stage (Nieuwkoop and Faber stage 10.5). Embryos were homogenized through a P200 pipet tip in 10 μl/embryo lysis buffer (20 mM Tris, pH 7.5, 1% Triton X-100, 140 mM NaCl, 10% glycerol, 10 mM EDTA, 1 mM DTT) supplemented with protease inhibitor cocktail (Sigma P8340, 1:100 dilution). Lysates were centrifuged at 1000 x g for 5 min to remove yolk protein, and subsequently at 14,000 rpm for 10 minutes to remove cellular debris. The lipid-free layer of supernatant was recovered and 1–2 embryo equivalents were loaded on to a 10% SDS-PAGE gel. Protein gels were run at 100 V and transferred to a nitrocellulose membrane for 2 h at 350 mA. Membranes were blocked for 1 h in bløk™-FL Fluorescent Blocker (Millipore), and probed with antibodies against β-tubulin (BD Pharmingen 556321, 1:1000), xWnt8 (see above, 0.14 μg/ml), Wnt1 (Abcam 15251, 1:1000), Wnt3a (Abcam 28472, 20 μg/ml) and Wnt5a (Cell Signaling Technology C27E8, 1:1000). Blots were incubated with fluorescently-conjugated secondary antibodies (LI-COR, 1:50,000) for 1 h, washed for at least 2 h (optimally overnight) in TBS-T, and visualized on a LI-COR Odyssey scanner.

Whole cell extracts were fractionated by SDS-PAGE and transferred to
nitrocellulose membrane by wet transfer for 4 h on ice at 60 V. Blots were
blocked in TBS blocking buffer (Li-Cor) and then stained overnight with
primary antibody at 4°C. The next day, membranes were washed three
times in TBST (TBS and 0.1% Tween-20), stained with fluorescently conjugated
secondary antibodies (Li-Cor) at 1:10,000 dilution in TBS blocking buffer
for 1 h at room temperature, and then imaged in the 680- and 800-nm
channels. Antibodies used: Gapdh (CST), Gpi1 (Thermo Fisher), Ldha (CST),
Pdha1 (Abcam), Tpi1 (Abcam), G6pdx (Abcam), a-tubulin (Santa Cruz).

Mouse liver, white adipose and gastrocnemius skeletal muscle was lysed in homogenization buffer. Protein concentrations were determined using the Bradford assay (Thermo Fisher) before separation by SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Licor) and blocked for 10 min in blocking buffer (Licor) diluted in TBST. Membranes were then incubated with Revert stain (Licor) per manufacturer’s instructions to quantitate total protein levels. Membranes were then incubated overnight with primary antibodies followed by a 60 min incubation at room temperature with fluorescently conjugated secondary antibodies (Licor). The following primary antibodies from Cell Signaling Technologies were used at a 1:1000 dilution: phosphorylated AKT (S473), phosphorylated GSK3β (S9) and GSK3β. The following primary antibodies from ProteinTech were used at a 1:1000 dilution: AKT, SOD1, SOD2 and catalase. Fluorescent intensity was quantified using Image Studio (Licor).