Anti β catenin

Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).

Soluble CD58 (sCD58), AKT inhibitor (LY294002), and Akt activator (SC-79) were acquired from Med Chem Express (MCE, Shanghai, China). Anti-Oct4 (11263-1-AP), anti-Sox2 (66411-1-Ig), anti-CD24 (18330-1-AP), anti-vimentin (10366-1-AP), and anti-c-Myc (10828-1-AP) were purchased from Proteintech (Wu Han, China). Anti-EPCAM (#2626S), anti-E-cadherin (#3195S), anti-AKT (#9272S), anti-GSK-3β (#5676S), anti-Phospho-GSK-3β (Ser9) (5558S), anti-β-Catenin (#8480S), anti-non-phosphorylated (active) β-catenin (S33/37/T41) (#8814S), anti-phospho-β-Catenin (Ser552) (#9566S), and anti-cyclin D1 (#55506S) were acquired from Cell Signaling Technology. Anti-CD58 (A0806) and anti-phospho-AKT (Ser473) (AP0140) were purchases from ABclonal (Wu Han, China).

Western Blotting was used to detect the influence of miR-410 and SLC34A2 on key proteins of Wnt/β-catenin signaling pathway. A549 or 95D cells were transfected with miR-410 mimics/NC or miR-410 inhibitors/NC, or transfected with p3.1-SLC34A2/p3.1 or siRNA-SLC34A2/siRNA-NC or cotransfected with siRNA-SLC34A2 and miR-410 inhibitors/NC according to the instructions of Lipo2000. The total membrane proteins were extracted 24hr post-transfection according to the manufacturer's instructions (Promega (Beijing) Biotech Co., Ltd, Beijing, China). Total cell protein was extracted using RIPA lysis buffer containing protease inhibitor cocktail at 1:100 dilution. Protein concentrations were measured using a BCA protein assay kit. The protein level was quantified by Western blotting analysis of 50 μg of cell extracts or tissue extracts. The following primary antibodies were used: anti-β-catenin (Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-DVL2 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-Gsk3β (Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-β-actin (Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-SLC34A2 (Santa Cruz, CA, USA, 1:500), β-actin was used as an internal control.

Western blots were performed as previously described [11] (link). Briefly, as soon as the harvested cells were lysed and the supernatant was collected, 60 µg protein was loaded onto SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). Membranes were blocked with 5% skimmed milk for 1 hour and incubated overnight with one of the following primary antibodies: anti-PSMD10 (diluted at 1∶1,000; Sigma, St. Louis, MO, USA), anti-Twist2 (diluted at 1∶200; Abcam, Cambridge, UK), anti-Vimentin (diluted at 1∶500; Cell Signaling Technology, Beverley, MA, USA), anti-β-catenin (diluted at 1∶500; Cell Signaling Technology), anti-E-cadherin (diluted at 1∶500; Cell Signaling Technology) and anti-cyclin D1(diluted at 1∶500; Cell Signaling Technology) rabbit monoclonal antibody, followed by 1 hour of incubation with the appropriate secondary antibody (1∶5,000). The anti-GAPDH (Epitomics) rabbit monoclonal antibody was diluted to 1∶1,000 for use as a sample loading control.

After washing with ice-cold PBS, cells were lysed with RIPA lysis buffer (Thermo Fisher) containing protease inhibitor (Roche). The protein concentration in the lysates was measured using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher), loading 20 μg of sample for each analysis. After separation in a 10% SDS-PAGE gel, proteins were transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% skim milk in Tris-buffered saline plus Tween (TBST). The blots were incubated at 4 °C overnight with the following primary antibodies: anti-LINGO-1 (1:500, Abcam), anti-Wnt5a (1:250, R&D Systems), anti-JNK (1:800, Santa Cruz), anti-p-JNK (1:800, Santa Cruz), anti-β-catenin (1:1000, Cell Signaling Technology), and anti-β-actin (1:2000, Cell Signaling Technology). After washing with TBST, the membranes were incubated for 1 h with the appropriate secondary antibody (1:2000, Abcam). The developed X-ray films were scanned and images were analyzed quantitatively with ImageJ software (version 1.45, NIH, USA). Relative protein levels were expressed as fold change after normalization to β-actin.