Phosphorylated ser473 akt primary antibody 4060t

Cell lysates were separated by 12% SDS-PAGE and transferred to the PVDF membrane. The membrane was then blocked with TBST buffer (TBS containing 0.1% Tween 20 and 5% non-fat dry milk) for 1 h, followed by an overnight incubation with primary antibody. AKT primary antibody (C67E7) and phosphorylated Ser473 AKT primary antibody (4060T) were purchased from Cell Signaling Technology Company. ERK primary antibody (AF1051), phosphorylated Thr202 ERK primary antibody (AF1891), and β-actin primary antibody (AF0003) were purchased from Beyotime Company. Axl primary antibody (AF8412) was purchased from Abbkine Company. Phosphorylated Tyr702 Axl primary antibody (AF8523) was purchased from Affinity Company. U2AF2 (4ab044957) and Gas6 primary antibody (4ab081330) were purchased from 4A Biotechnology Company. After being extensively washed, the membranes were incubated with HRP-labeled secondary antibodies at room temperature for 2 h. Finally, the membranes were washed 4 times with TBST and visualized by BeyoECL Moon kit (Beyotime, China), imaging was then performed using a biochemi-luminometer (General Electric, AI600). Grayscale quantifications of bands were performed using Image J software (National Institutes of Health, USA).

The cells were lysed with RIPA buffer, and then the protein concentration was detected by BCA protein detection kit (Beyotime, China). 20 µg of whole-cell lysates was separated by SDS-PAGE and then transferred to a pre-activated Polyvinylidene Fluoride membrane (PVDF), the membrane was blocked for 1 h in TBST buffer (TBS containing 0.1% Tween 20) containing 5% non-fat dry milk followed by an overnight incubation with primary antibody diluted. AKT primary antibody (C67E7) and phosphorylated Ser473 AKT primary antibody (4060 T) were purchased from Cell Signaling Technology Company. ERK primary antibody (AF1051), phosphorylated Thr202 ERK primary antibody (AF1891) and β-actin primary antibody (AF0003) were purchased from Beyotime Company. SRSF1 primary antibody (NHA3445) was purchased from Novogene Company. After extensive washing, the blot was incubated with a secondary antibody overnight. Finally, the membranes were washed thrice with TBST and visualized by BeyoECL Moon kit (Beyotime, China). Imaging was then performed using a biochemi-luminometer (General Electric, AI600) and Image J software was ultized to quantify the bands’ grayscale.