Antibodies against phospho akt ser473

Pentamidine was obtained from MedChemExpress (Shanghai China). Cycloheximide (CHX) was obtained from Sigma-Aldrich (St. Louis, MO, USA). MG132 was obtained from Cell Signaling Technology (Beverly, MA, USA).
Antibodies against Phospho-AKT (Ser473, Cat. #. 4060T, 1:1000), AKT (Cat. #. 4691T, 1:1000), E-Cadherin (Cat. #. 3195T, 1:1000) and snail (Cat. #. 3879P, 1:1000) were obtained from Cell Signaling Technology. Antibodies against PTEN (Cat. #. 22034-1-AP, 1:2000), N-Cadherin (Cat. #. 62219-1-lg, 1:2000), GAPDH (Cat. #. 60004-1-lg, 1:20,000) and ubiquitin (Cat. #. 10201-2-AP, 1:500) were obtained from Proteintech.

Western blotting was conducted after separation of polypeptides using SDS-PAGE. Proteins on gel were transferred to PVDF membrane (Merck Millipore; Billerica, MA, USA). The membrane was further incubated with indicated primary and appropriate secondary antibodies. Antibodies against ACOX1 were obtained from Proteintech Group (Chicago, IL, USA). Phosphorylated form of ERK1/2 and ERK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and BD Biosciences, respectively. Antibodies targeting MMP3, MMP10 and phospho-Akt (Thr308) were also purchased from Santa Cruz Biotechnology. Antibodies against phospho-Akt (Ser473) were obtained from Cell Signaling Technology (Danvers, MA, USA). MMP2 and MMP9 antibodies were purchased from Abcam (Cambridge, Cambridgeshire, UK). An anti-GAPDH antibody (BioWorld; St. Louis Park, MN, USA) was used as control. HRP-conjugated secondary antibodies against rabbit IgG or mouse IgG (GeneTex) were incubated with membrane for 1 h at room temperature. Immunobands were detected using chemiluminescent HRP substrates (ECL; Merck Millipore) and captured by UVP BioSpectrum 600 Imaging System. The intensity of bands was quantified by Image J software.