Y409094

Target tissue samples or cells were homogenized in ice-cold RIPA lysis buffer containing 1% protease inhibitor for 30 min. The lysates were centrifuged, and the supernatants were then recovered. The protein concentrations were measured with a bicinchoninic acid protein assay kit. The protein samples were then applied for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene fluoride membrane. The membranes were firstly blocked with 5% non-fat milk and then hybridized with the following antibodies: α-SMA (55135-1-AP; Proteintech, Wuhan, China), collagen I (66761-1-Ig, Proteintech), E-cadherin (20874-1-AP, Proteintech), vimentin (10366-1-AP, Proteintech), OGN (A07061, Boster, Pleasanton, CA, USA), p-mTOR (ab109268, Abcam), mTOR (ab2732, Abcam), Akt (Y409094, ABM, New York, NY, USA), p-Akt (Y011054, ABM) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies and ECL were used for detection and the protein expression was normalized to that of β-actin.

Fresh tumor tissues from nude mice were fixed in formalin at room temperature for 24 h; then, the tissues were dehydrated with an ethanol and acetone series, embedded in paraffin, and sectioned onto glass slides at 4 μm. The slides were stained with hematoxylin and eosin (H&E) following deparaffinization. For IHC (immunohistochemistry), the sections were incubated at 65°C for 2 h and hydrated in xylene and graded ethanol. Then, the sections were immersed in a citrate buffer solution, heated in a microwave oven for 2.5 min, kept 5 min at room temperature, and heated in a microwave oven for 24 min. After washing with PBS (Phosphate Buffer Solution) three times, the slides were incubated with a blocking buffer for 10 min and then with a primary antibody overnight at 4°C. Primary antibodies include a rabbit antibody against vimentin (1:5,000; Proteintech, 10366-1-AP), the E-cadherin rabbit polyclonal antibody (1:1000; Proteintech, Wuhan, China, 20874-1-AP), p-AKT antibody (1:50; Y011054, abm, Richmond, BC, Canada), and AKT (1:200; Y409094, abm, Richmond, BC, Canada). After washing with Phosphate Buffer Solution, the sections were incubated with a secondary antibody, detected with DAB (diaminobenzidine), and counterstained with hematoxylin. Positive staining was deemed as brown dots.

Extract total protein from target cells, separate the total protein samples using SDS-PAGE, and transfer the protein samples to polyvinylidene fluoride membranes. For the blockage of nonspecific binding, the membranes were incubated with 5% skim milk dissolved in diphenyltris buffer saline for 2 h at room temperature. Incubate the membranes with the primary antibodies against RNF139 (Catalog # MBS421811; MyBioSource, Inc., San Diego, CA, USA), PI3K (AF6241; Affinity, Cincinnati, OH), p-PI3K (AF3242, Affinity), Akt (Y409094; ABM, Richmond, Canada), or p-Akt (Y011054, ABM) overnight at 4 °C. The incubation with the primary antibodies was followed by another incubation with the corresponding horseradish peroxidase coupled secondary antibody for 2 h at room temperature. Finally, the enhanced chemiluminescence visualization scan was performed.