Western Blot Analysis of Tight Junctions

Western blot analysis was performed as previously reported,³⁰ (link) with minor modifications. In brief, HRMECs were treated with 10-nM SZN-413-p 24 hours before harvesting for cell lysis. The anti-ZO-1 (ab216880; Abcam, Cambridge, UK), anti-CLDN5 (ab131259; Abcam), and anti-β actin (A5441; Sigma-Aldrich, St. Louis, MO) primary antibodies were used, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for chemiluminescent signals.

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Nguyen H., Chen H., Vuppalapaty M., Whisler E., Logas K.R., Sampathkumar P., Fletcher R.B., Sura A., Suen N., Gupta S., Lopez T., Ye J., Tu S., Bolaki M., Yeh W.C., Li Y, & Lee S.J. (2022). SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy. Translational Vision Science & Technology, 11(9), 19.

Publication 2022

ab216880 ab131259 anti-β actin

Actin Anti antibodies Antibodies Cell Horseradish peroxidase Mouse Rabbit Western blot analysis

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Variable analysis

independent variables

Treatment with 10-nM SZN-413-p

dependent variables

Levels of ZO-1 protein
Levels of CLDN5 protein
Levels of β-actin protein

control variables

Harvesting time (24 hours after treatment)

controls

Positive control: Not specified
Negative control: Not specified

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