E bc r327

Western blot assay was proceeded based on the previous study.¹⁸ Simply put, total protein of LX‐2 cells was extracted with RIPA lysis buffer (E‐BC‐R327; Elabscience). After the protein concentrations were measured by BCA protein assay, 30 μg of the protein was separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels, and transferred onto polyvinylidene fluoride membranes (3010040001; Roche). Then, the membranes were blocked, followed by the incubation with primary antibodies at 4°C overnight and a secondary antibody at 37°C for 1 h. The protein band was exposed with ECL buffer (R30199; Pierce), and the intensity analysis was achieved with iBright FL1500 Imaging System (A44115; Invitrogen). The primary antibodies used were those against G6Pase (1:1000; Rabbit; ab93857; abcam, 40 kDa), PEPCK (1:1000; Rabbit; No. ABIN2855891; antibodies‐online, 71 kDa), p‐PI3K (1:1000; Rabbit; #029801; Alamo Laboratories Inc, 85 kDa), PI3K (1:1000; Rabbit; #4249; Cell Signaling Technology (CST), 110 kDa), p‐Akt (1:2000; Rabbit; #4060; CST, 60 kDa), Akt (1:1000; Rabbit; #4685; CST, 60 kDa), insulin‐like growth factor‐1 receptor (IGF‐1R, 1:1000; Rabbit; ab182408; abcam, 156 kDa) and GAPDH (1:10000; Rabbit; ab181602; abcam, 36 kDa). The secondary antibody was goat anti‐rabbit antibody (1:2000, #14708; CST). GAPDH was served as a loading control.

Total protein was prepared by extracting the supernatant after lysing tissues or cells with RIPA lysis buffer (Elabscience, E‐BC‐R327) and subsequently adding a loading buffer (wshtbio, ES003). The proteins were resolved using a 12.5% SDS‐PAGE gel (Epizyme, Shanghai, China) and subsequently blotted onto 0.22 μm nitrocellulose membranes (Millipore, America). After incubation with 5% BSA for 1 hour at room temperature, the membrane was incubated with primary antibody overnight at 4°C. The primary antibodies used were as follows: anti‐GAPDH (1:1000; Elabscience, E‐AB‐40337), anti‐CCR6 (1:1000; SinoBiogical, #106771‐T32), anti‐IL‐17A (1:1000; Affinity, DF6127), anti‐CCL20 (1:1000; Affinity, DF2238), anti‐P65 (1:1000, Affinity, AF5006), anti‐Phospho‐P65 (1:1000, Affinity, AF2006), anti‐ERK1/2 (AF0155), anti‐Phospho‐ERK1/2 (1:1000, Affinity, AF1015), anti‐P38 (1:1000, Affinity, AF6456), anti‐Phospho‐P38 (1:1000, Affinity, AF4001), anti‐mTOR (1:1000, CST, 2983 T), anti‐Phospho‐mTOR (1:1000, CST, 5536 T), anti‐AKT (1:1000, CST, 4691 T) and anti‐Phospho‐AKT (1:1000, CST, 4060 T). Then the bands were incubated with the secondary antibodies (1:5000; Elabscience, E‐AB‐1003), which was derived from rabbit and conjugated with HRP. Subsequently, blots were captured with an ECL luminescence system (ClinX, Shanghai, China). The gels were quantified and analysed using ImageJ.

After washing twice with cold PBS, TMCs were lysed in RIPA buffer (cat. no. E-BC-R327; Elabscience, Wuhan, China) to extract total protein. The protein samples (30 μg) were denatured and separated by 10% SDS/PAGE. Protein was then moved onto PVDF membranes and blocked with 5% skimmed milk containing PBS to block nonspecific binding. Subsequently, the membranes were cultured overnight at 4°C with primary detection antibodies, including cleaved caspase-3 (ab32042; Abcam, Cambridge, USA), cleaved caspase-9 (# 20750S, Cell Signaling Technology, MA, USA), collagen I (ab138492), fibronectin (ab268020), laminin (ab108536), SMAD2 (ab40855), TGF-β (ab215715), and β-actin (# 4970S; Cell Signaling Technology). Appropriate secondary antibodies were then incubated with the blots for 1 h at room temperature. After washing, the signals were monitored with ECL Advance reagents (GE Healthcare, Braunschweig, Germany) and analyzed with Image Lab v6.0 software.