Anti p gsk3β ser9

The antibodies used: anti-ASIC1a, anti-Bcl-2, anti-cleaved-caspase3, anti-Bax, anti-c-Myc, anti-Cyclin D, anti-CDK4, anti-GSK3β, anti-p-GSK3β (Ser9), anti-β-catenin, anti-p-β-catenin (Ser33) were from Abcam (Cambridge, UK). Anti-GAPDH, anti-HA and anti-Lamin B were from Santa Cruz Biotechnique (Santa Cruz, USA). Dual luciferase reporter assay system was from Promega Corporation (Wisconsin, USA). Reporter constructs were generated by incorporating 8X lymphocyte enhancer-binding factor-T cell factor (LEF-TCF) consensus binding sites into the pGreenFire1 (pGF1) vector containing eGFP-T2A-lucifersase as the reporter (System Biosciences) [29 ]. cDNAs were cloned into mammalian expression vectors pCDNA6-CMV-V5/His (Invitrogen) or a modified pCDH1-EF1 vector (System Biosciences). We obtained from Addgene the pcDNA3-HA-Ub construct (no. 18712) [30 (link)]. Lenti-cas9 and Lenti-sgRNA were purchased from Genechem (Shanghai, CHN). Cells were firstly infected with Lenti-cas9 and selected by puromycin. The stable sub-lines were then infected with Lenti-sgRNA to specifically knockout target genes. The sgRNA used were sg-GFP: 5′- GGTGAACCGCATCGAGCTGA-3′; sg-ASIC1a: 5′- GACGAGACGTCCTTCGAAGC-3′. All other chemicals were from Sigma–Aldrich Corporation (San Luis, USA) unless otherwise stated.
ASIC1α-specific inhibitor PcTx1 was purchased from Abcam (ab120483, Cambridge, MA, USA).

Total proteins of cell samples were extracted with lysis buffer and then quantified using the BCA method (KeyGen Biotech, Jiangsu, China). The lysate was diluted in SDS sample buffer (KeyGen Biotech) for SDS-polyacrylamide gelelectrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences,USA). The membranes were immunoblotted at 4 °C overnight with anti-PRDX2 (Proteintech, USA), anti-c-Myc(Abcam, UK), anti-p-c-Myc(S62) (Abcam), anti-p-c-Myc(T58)(Abcam), anti-E-cadherin (Proteintech), anti-Vimentin (Proteintech), anti-N-cadherin (Proteintech), anti-GSK3β (Abcam), anti-p-GSK3β (Ser9) (Abcam), anti-AKT(1/2) (Abcam), anti-AKT1 (Abcam), anti-AKT2 (Abcam), anti-p-AKT2 (Ser474) (Abcam) and anti-GAPDH (Proteintech) antibodies at appropriate dilution concentration, followed by incubation using the appropriate second antibodies for 2 h. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Image J software was used to analyze the grey value of the interest protein.

Standard Western blot assays were used to analyze protein expression in small intestinal tissues. Briefly, tissues were lysed using the lysis buffer (10Mm Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₄, 2 mM Na₃VO₄, 1% Triton X-100, 10% Glycerol, 0.1% SDS, 0.5% Deoxycholate Acid). Protein lysates were separated on a 4 to 20% Tris-glycine gel and transferred to a polyvinylidene difluoride membrane. The following antibodies were used: anti-AKT (Santa Cruz, 1:2000), anti-p-AKT-Ser473 (Cell Signaling, 1:2000), anti-GSK3β (Cell signaling, 1:1000), anti-p-GSK3β-Ser9 (Abcam, 1:2000) and anti-β-actin (Sigma, 1:100000) antibodies.