Western blot

The antibodies used for the immunoblot analysis are as follows: anti-pERK(1/2) (4377T), anti-ERK(1/2) (9102S), and anti–glyceraldehyde phosphate dehydrogenase (2118S) from Cell Signaling Technology. 4G10 was from Millipore (05-321), and anti-GFP (ab290) was from Abcam. Briefly, the cells were stimulated as indicated, and then the cells were lysed using radioimmunoprecipitation assay buffer (9806S) on ice for 10 min and then centrifuged at 4°C and 13,000 rpm. The protein concentration was measured using a Bio-Rad protein assay dye reagent (Bio-Rad, #5000006) following the protocol of the manufacturer. The same amount of boiled proteins was loaded into precast 4 to 20% gradient SDS–polyacrylamide gel electrophoresis (PAGE) (cat#4561096) or 10% precast SDS-PAGE gel for ERK phosphorylation measurement. The gel was run at 110v for 60 to 90 min and then transferred to a nitrocellulose filter membrane at 110v for 60 min. Dry milk (5%) was used to block the membrane at room temperature for 30 min. Then, the membrane was incubated with primary antibody at 4°C overnight, followed by incubation with the secondary antibody at room temperature for 1 hour. Last, the membrane was visualized with standard chemiluminescence.