Secondary antibodies of the horseradish peroxidase hrp conjugated goat anti rabbit igg

Vascular endothelial growth factor was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against phosphor-VEGFR2 (p-VEGFR2, Y1175), VEGFR2, Akt, p-Akt (S473), Caspase (cysteine-aspartic proteases) 9, and p-BAD (Cell Signaling Technology; Danvers, MA, USA). Aflibercept (40 μg/μL) and ranibizumab (10 μg/μL) were from the pharmacy of Massachusetts Eye and Ear (Boston, MA, USA). The primary antibody against β-actin and secondary antibodies of the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescent substrate for detection of HRP was obtained from Thermo Fisher Scientific (Waltham, MA, USA).

VEGF was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against VEGFR2, Akt, and p-Akt (S473) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibody against β-actin and secondary antibodies of the horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescent substrate for detection of HRP was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The antibodies against p53 and MDM2 were purchased from Cell Signaling Technology (Danvers, MA, USA) and from Abgent (San Diego, CA, USA), respectively. The primary antibody against β-actin and the secondary antibodies of the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescent substrate for detection of HRP was from Thermo Scientific (Waltham, MA, USA).
hRPE cells were purchased from Lonza (Walkersville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 medium (F12) (Thermo Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The hRPE cells with MDM2 T309G or T309T only were created using a system of AAV-SpCas9 D10A and AAV-SpGuide (MDM2 or lacZ) and a homology direct recombinant DNA template with MDM2 G309 as described previously.^{21 (link)} All cells were cultured at 37°C in a humidified 5% CO₂ atmosphere.