Rabbit anti erk

Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum were purchased from Hyclone (Logan, UT, USA). Lipofectamine 2000 and TRIzol Reagent were obtained from Invitrogen (Carlsbad, CA, USA). M-MLV reverse transcriptase was purchased from Promega (Madison, WI, USA). The antibodies used were as follows: rabbit anti-GPR137 (Proteintech Group, Chicago, IL, USA), rabbit anti-phospho-AMP-activated protein kinase (AMPK)α (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-proline-rich AKT substrate of 40 kDa (PRAS40) (Cell Signaling Technology), rabbit anti-phospho-extracellular signal-regulated kinase (ERK)1/2 (Cell Signaling Technology), rabbit anti-AKT (Proteintech Group), rabbit anti-phospho-AKT (Cell Signaling Technology), rabbit anti-ERK (Proteintech Group), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (Proteintech Group), and goat anti-rabbit horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Lentiviral vector pFH-L and the lentiviral packaging auxiliary carriers pVSVG-I and pCMV△R8.92 were purchased from Duruobio (Shanghai, China). SYBR Green Master Mix Kits and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cells were lysed with RIPA buffer and centrifuged at 13,000 g for 10 min. Protein lysates were separated using SDS/PAGE, blotted onto a PVDF membrane, and probed overnight at 4°C with antibodies against rabbit anti‐SHCBP1 (1:1000; Sigma), mouse anti‐GAPDH (1:1000, Proteintech) or rabbit anti‐BCL‐2 (1:1000; Proteintech), rabbit anti‐caspase 3 (1:1000; Abcam), rabbit anti‐caspase 3 cleave (1:1000; Abcam), rabbit anti‐caspase 9 (1:1000; Abcam), rabbit anti‐E‐cadherin (1:1000; Cell Signaling Technology), rabbit anti‐vimentin (1:1000; Cell Signaling Technology), rabbit anti‐AKT (1:1000; Cell Signaling Technology), rabbit anti‐p‐AKT (1:1000; Cell Signaling Technology), rabbit anti‐ERK (1:1000; Proteintech), rabbit anti‐p‐ERK (1:1000; Cell Signaling Technology), rabbit anti‐PI3K (1:1000; Cell Signaling Technology), rabbit anti‐p‐PI3K (1:1000; Cell Signaling Technology). Stripped membranes were probed with a secondary antibody of goat anti‐mouse or anti‐rabbit IgG (1:10,000, Bioworld) and then visualized with enhanced chemiluminescence.