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. 2023 Oct 9;24(19):15032. doi: 10.3390/ijms241915032

Figure 4.

Figure 4

Fluorescent Western blotting for cell adhesion molecules (VCAM1 and ICAM1), transcription factors of endothelial-to-mesenchymal transition (Snail and Slug, TWIST1, and ZEB1), mechanosensitive transcription factors (KLF2, KLF4, and NRF2), endothelial nitric oxide synthase eNOS, and loading control (GAPDH and CD31) in human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) cultured at static conditions. (A) VCAM1 (pro-inflammatory cell adhesion molecule, green)/GAPDH (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (B) ICAM1 (pro-inflammatory cell adhesion molecule, green)/CD31 (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (C) Snail and Slug (endothelial-to-mesenchymal transition transcription factor, green)/CD31 (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (D) TWIST1 (endothelial-to-mesenchymal transition transcription factor, green)/ZEB1 (another endothelial-to-mesenchymal transition transcription factor, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (E) TWIST1 (endothelial-to-mesenchymal transition transcription factor, green)/KLF2 (atheroprotective mechanosensitive transcription factor, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (F) KLF4 (atheroprotective mechanosensitive transcription factor, green)/eNOS (endothelial nitric oxide synthase, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom); (G) NRF2 (atheroprotective mechanosensitive transcription factor, green)/GAPDH (loading control, red), fluorescent Western blot (top), and total protein staining confirming an equal protein loading (bottom). Each band within the groups represent a protein lysate from one experiment (n = 3 experiments in total). Total protein normalisation was conducted by Fast Green FCF staining of the membranes after the fluorescent imaging to ensure the equal protein loading at all blots (in addition to loading controls such as GAPDH or CD31). Fluorescent ladder (L) and molecular weight signatures (kDa) are provided to the left of the HCAECs and HITAECs protein bands. Ratios of 1:100 and 1:200 are dilutions of the antibody against TWIST1, highlighted to show low expression of this protein in the quiescent ECs.