α tubulin

Cells were collected and lysed with RIPA lysis buffer (Co Win Biosciences, China) according to the manufacturer’s instructions, and proteins were harvested. Protein quantification was performed using a BCA Protein Quantification Kit (Thermo, USA), and samples were adjusted to the same concentration. After boiling in 6 × Loading Buffer for 5 min, the proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membrane was incubated overnight at 4 °C with the appropriate primary detection antibodies: GAPDH (Sigma, 1:1,000), α-Tubulin (Sigma, 1:600), Snail (Cell Signaling Technology, 1:1,000), and Vimentin (Cell Signaling Technology, 1:1,000); AKT (ProteinTech, 1:1,000) and p-AKT (ProteinTech, 1:1,000). After washing three times with TBST for 5 min, the membrane was incubated with secondary antibodies (Cell Signaling Technology, 1:1,000) for 1 h at 37 °C. After washing three times with TBST for 15 min each time, the protein bands were visualized by fluorography using an enhanced chemiluminescence system (Millipore, USA); the expression levels of α-Tubulin and GAPDH were used as internal references.

Spinal cords were isolated from control and mutant E18.5 embryos. Tissue lysates were prepared and diluted to protein concentration of 0.4 μg/μl using sample preparation kit (ProteinSimple). Tissue lysates were examined using automated western blot system, WES System (ProteinSimple) as per manufacturer’s protocol. The following primary antibodies were used for analysis: ZPR1 (Clone LG-C61), Flag M2 (#F1804, Sigma), HoxA5, HoxC5, α-tubulin (#T8203, Sigma) and β-Actin (#A5316, Sigma). Data analysis and quantitation of protein levels were performed using Compass Software (Protein Simple). For standard immunoblot method, cell lysates were prepared from control and transfected HeLa cells using Triton lysis buffer^{5 (link)}. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane (Millipore). Proteins were detected using primary antibodies (1:100), ZPR1 (Clone LG-C61), Flag M2 and α-tubulin (#T6074, Sigma) followed by HRP-conjugated donkey anti-mouse or rabbit IgG (1:5000) secondary antibodies. Chemiluminescence and quantitation of immunoblots was performed using ImageQuant LAS4000. The relative levels of proteins (mean ± s.e.m.) normalized to either actin or tubulin, are presented as bar graphs.