Anti p β catenin

Ovaries were collected and homogenized in lysis buffer and protease inhibitor cocktai containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 2 mg/ml aprotinin, and 5 mM ethyl glycol tetraacetic acid (EGTA, Beyotime Biotechnology, China). Then the tissues were centrifuged at 12,000× g for 10 min to obtain supernatants which were diluted to 1 µg/µl with 4 × sample buffer and frozen at − 20 °C. Proteins were quantified with the Lowry Assay. The tissue extracts were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked and incubated with primary antibodies of anti-β-actin (1:1000, Cell Signaling, USA), anti-β-catenin (1:1000, Cell Signaling, USA), anti-p-β-catenin (1:1000, Cell Signaling, USA) and anti-GSK-3β (1:1000, Cell Signaling, USA) at 4 °C overnight, then reacted with a secondary antibodies of anti-rabbit IgG (1:1000, Cell Signaling, USA) after removing unconjugated antibodies by tris-buffered saline Tween-20 (TBST). The peroxidase activity was visualized using an enhanced chemiluminescence (ECL) kit (Biosharp, USA) to image the proteins recognized by the antibodies.

Anti-FERMT3 (ab68040), anti-E-cadherin (ab40772), anti-Vimentin (ab92547) and anti-Snail (ab216347) were purchased from Abcam (Abcam, USA). Anti-GAPDH (AB0037) was purchased from Abways Biotechnology (Shanghai, China). Anti-β-catenin (#8480), anti-p-β-catenin (#5651), anti-GSK-3β (#12456), anti-GSK-3β (#5558) was purchased from Cell Signaling Technology. Lithium Chloride (LiCl), an activator of the Wnt/β-catenin signaling pathway, was purchased from Sigma Aldrich.

Tissues and cells were homogenized. Solubilized proteins in lysis buffer (0.1 M Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 12% 2‐mercaptoethanol, and BPB) were subjected to SDS–PAGE, and proteins were electrotransfered to nitrocellulose membranes. Immunodetection was carried out using an ECL kit (GE Healthcare) according to the manufacturer's protocol. Antibodies used were as follows: anti‐β‐catenin (1:5,000, BD, #610154), anti‐P‐β‐catenin (1:2,000, Cell Signaling Technology, #4176), anti‐active‐β‐catenin (1:2,000, Cell Signaling Technology, #19807), anti‐PTEN (1:2,000, Cell Signaling Technology, #9559), anti‐P‐PTEN (1:2,000, Cell Signaling Technology, #9554), anti‐AKT (1:1,000, Cell Signaling Technology, #9272), anti‐P‐AKT (1:1,000, Cell Signaling Technology, #9271), anti‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #3018), anti‐P‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #4568), and anti‐HSC70 (1:2,000, Santa Cruz Biotechnology, #sc7298).

Western blot was performed as previous study described [9 (link)] using anti-GSK3β, anti-cyclinD1 (BD Pharmingen, Franklin Lakes, NJ, USA), anti-p-β-catenin, anti-β-catenin (Cell Signaling Technology,Danvers, MA,USA), anti-c-Myc and anti-MMP7 (Bioworld Technology, St. Louis Park, MN, USA). An anti-α-tubulin monoclonal antibody (Sigma, St. Louis, MO, USA) was used as a loading control [9 (link)].

Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, China). An NE-PER™ Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific, MA) was used according to the manufacturer’s instructions for extracting nuclear and cytoplasmic proteins from the different groups.
A 12% sodium dodecyl sulfate‐polyacrylamide gel was chosen for total protein separation, and the proteins were then transferred to nitrocellulose membranes (Millipore, USA). The membranes were incubated with primary antibodies, including anti-LC3B (Abcam Cat# ab192890, RRID: AB_2827794), anti-P62/SQSTM1 (Abcam Cat# ab207305, RRID: AB_2885112), anti-β-actin (Proteintech# 20536-1-AP), anti-LDHA (Cell Signaling Technology Cat# 3582, RRID: AB_2066887), anti-β-catenin (Cell Signaling Technology Cat# 8480), anti-p-β-catenin (Cell Signaling Technology Cat# 4176), anti-c-Myc (Covance Cat# MMS-150P-1000, RRID: AB_291322), anti-GSK3-β (Cell Signaling Technology Cat# 121456), anti-Axin1 (Cell Signaling Technology Cat# 2087, RRID: AB_2274550) and anti-Histone H3 (Cell Signaling Technology Cat# 4499, RRID: AB_10544537). Enhanced chemiluminescence reagents (Millipore, USA) were used to assess protein expression.