Western blots were performed using standard protocols. Immediately after dissection, twelve hyaloid vascular tissues or retinae of P6 (six pups) were pooled in 100 μl of 1x Laemmli sample buffer and sonicated. After centrifugation, 20 μl of supernatant was loaded on 4 to 20% gradient protein gel (ThermoFisher Scientific). Separated protein bands were transferred to PVDF membrane, and bands were visualized by chemiluminescence (ThermoFisher Scientific). Unprocessed immunoblots are available in Supplementary Fig. 6 . Band intensity was measured by ImageJ (NIH). Following antibodies were used for Western blotting: VEGFR2 (Cell Signaling Technology, #9698), phospho-VEGFR2 (Cell Signaling Technology, #2478), β-tubulin (Abcam, ab6046), DAT (Novus, NB300–254), phospho-DAT (ThermoFisher Scientific, PA5–35414), AKT (Cell Signaling Technology, #4691) and phospho-AKT S473 (Cell Signaling Technology, #4060). All antibodies were used at 1:1000 dilution.
4 to 20 gradient protein gel
Manufactured by Thermo Fisher Scientific
The 4 to 20% gradient protein gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It features a continuous gradient of polyacrylamide concentration, allowing for the effective separation of a wide range of protein sizes in a single electrophoresis run.
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2 protocols using 4 to 20 gradient protein gel
1
Western Blot Analysis of Vascular Factors
2
Western Blot Analysis of Vascular Factors
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Western blots were performed using standard protocols. Immediately after dissection, twelve hyaloid vascular tissues or retinae of P6 (six pups) were pooled in 100 μl of 1x Laemmli sample buffer and sonicated. After centrifugation, 20 μl of supernatant was loaded on 4 to 20% gradient protein gel (ThermoFisher Scientific). Separated protein bands were transferred to PVDF membrane, and bands were visualized by chemiluminescence (ThermoFisher Scientific). Unprocessed immunoblots are available in Supplementary Fig. 6 . Band intensity was measured by ImageJ (NIH). Following antibodies were used for Western blotting: VEGFR2 (Cell Signaling Technology, #9698), phospho-VEGFR2 (Cell Signaling Technology, #2478), β-tubulin (Abcam, ab6046), DAT (Novus, NB300–254), phospho-DAT (ThermoFisher Scientific, PA5–35414), AKT (Cell Signaling Technology, #4691) and phospho-AKT S473 (Cell Signaling Technology, #4060). All antibodies were used at 1:1000 dilution.
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