Goat anti rabbit secondary antibody

All cells were washed with PBS for twice and lysed in IP lysis buffer (Beyotime, China) supplemented with protease and phosphatase inhibitor on ice for 30 min. After that, cell lysates were centrifuged for 30 min (12000 g, 4 °C). Then the protein sample was collected and the protein concentration was measured by using BCA protein concentration assay kit (Beyotime, China). Protein samples were diluted to a certain concentration (5 ug/ul) with the lysis buffer. The proteins (50 μg) were separated by 12%SDS-PAGE and transferred to PVDF membranes (Millipore). After being blocked with skimmed milk, the blots were incubated overnight at 4°C with rabbit Anti-Foxa2 and anti-β-actin. The membranes were then washed and incubated with goat anti-rabbit secondary antibodies for 60 min, and then developed with BeyoECL Plus kit (Beyotime). The antibodies were purchased from the following sources: Anti-Foxa2 (1:1000, Cell Signaling Technology #3143, USA), anti-β-actin (1:1200, Cell Signaling Technology #8457, USA) and goat anti-rabbit secondary antibodies (1:1200, Santa Cruz, sc2030, USA).

The rats were euthanized and the superficial dorsal horn (lumbar 4-6) tissues were dissected under an anatomical microscope. The tissues were then homogenized in ice-cold radioimmunoprecipitation assay buffer containing 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail kit (Sigma-Aldrich, St. Louis, MO). Then the tissues were centrifuged. The supernatant was then diluted to the same volume and applied to SDS-PAGE. Membranes were incubated with the rabbit anti-p-mTOR/p-S6K1/p-4EBP1antibodies; rabbit anti-mTOR /S6K1/4EBP1 antibodies; rabbit anti-p-PI3K p85/ anti- PI3K p85, respectively. All these primary antibodies were purchased from the Abcam Company (Cambridge, UK); and goat antirabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Immunoreactive proteins were detected by enhanced chemiluminescence. The membrane was also processed to detect β-actin for equal loading. The optical density of protein bands was first analyzed using the NIH Scion image Software ImageJ (http://rsb. info.nih.gov/ij/), and values for densities of immunoreactive bands/β-actin band densities from the same lane were determined. Each of the values was then normalized to a control sample.