Hrp conjugated against rat or rabbit

Lysis was done with RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing protease and phosphatase inhibitors (Complete ULTRA Tablets, Roche, Nutley, NJ, USA). After centrifugation, the supernatant was collected and quantified by BCA assay (Sigma-Aldrich). The proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk, the membranes were probed with rabbit anti-VEGF₁₆₅ (R&D System, Los Angeles, CA, USA), rabbit anti- VEGF₁₆₅b (R&D System), rabbit anti-SRPK1 (Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (Cell Signaling Technology), rabbit anti-cleaved caspase 3 (casps3; Cell Signaling Technology), rabbit anti-CCND1 (Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-CDK4 (Santa Cruz Biotechnology), rabbit anti-nitric oxide synthase (anti-NOS3; Cell Signaling Technology), and rabbit anti-α-tubulin (Cell Signaling Technology, Danvers, MA, USA). Secondary antibodies are HRP-conjugated against rat or rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA). The protein levels were first normalized to α-tubulin, and then normalized to the experimental controls. Densitometry of Western blots was quantified with NIH ImageJ software.