Phosph erk1 2

The target cells and mice choroids were harvested at various time points according to the experiment design. Each choroid protein sample in the different groups was extracted from 10 eyeballs. First, the antibodies specific for VEGF-A, total ERK 1/2, phosph-ERK 1/2, AKT, phosph-AKT and cleaved caspase-3 and β-actin (Cell Signaling Technology, Inc., Boston, MA, USA) were incubated at 4 °C overnight with dilutions of 1:1000. The polyvinylidene fluoride (PVDF) membranes were washed with a mixture of tris-buffered saline and Polysorbate 20 (TBST), three times. Then, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (1:5000) for an additional 1 h. Finally, enhanced chemiluminescence was used to detect the immune complexes. All data were repeated at least three times.

Cells and tissue samples were collected and incubated in the RIPA buffer (Sigma-Aldrich) to obtain proteins. Approximately 30 μg of total proteins were loaded into each lane of 10-15% SDS-PAGE, separated in electrophoresis at a constant pressure of 160 V for about 1-2 h, and transferred onto nitrocellulose membranes (Millipore). After blocking, the membranes were then incubated with primary antibodies against EGFR (Santa Cruz Biotechnology), phosph-EGFR (Santa Cruz Biotechnology), HER2 (Santa Cruz Biotechnology), RAS (Santa Cruz Biotechnology), RAF1 (Santa Cruz Biotechnology), MEK1/2 (Cell Signaling), phosph-MEK1/2 (Cell Signaling), ERK1/2 (Cell Signaling), phosph-ERK1/2 (Cell Signaling), and β-actin (Santa Cruz Biotechnology). β-actin was loaded as an internal reference. Bands were then treated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). Bands were developed using chemiluminescence substance (Thermo Scientific). All experiments were conducted three times independently with similar results. A representative result for Western blots was provided.

As previously described [20 (link)], cells were harvested in RIPA buffer (Sigma Aldrich). Whole cell lysate was separated using SDS-PAGE. Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.