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. Author manuscript; available in PMC: 2025 Apr 27.
Published in final edited form as: Methods Mol Biol. 2024;2711:235–240. doi: 10.1007/978-1-0716-3429-5_19

Determination of tight junction integrity in brain endothelial cells based on tight junction protein expression

Himakarnika Alluri 1, Chander Sekhar Peddaboina 2, Binu Tharakan 3
PMCID: PMC12034388  NIHMSID: NIHMS2067570  PMID: 37776462

Summary:

Blood-brain barrier (BBB) dysfunction and hyperpermeability that occurs following traumatic and ischemic insults lead to various downstream ill effects such as cerebral edema and elevation of intracranial pressure. The inter-endothelial tight junctions that consist of tight junction proteins are critical regulators of BBB dysfunctions and hyperpermeability. The major tight junction-associated proteins of the BBB are occludin, claudins, and junctional adhesion molecules that are intracellularly linked to the adaptor protein zonula occludens-1 (ZO-1). Quantitative measurement of tight junction-associated proteins provides valuable insight into barrier integrity and mechanisms that regulate microvascular hyperpermeability. Western blot analysis is a commonly used method to separate and identify proteins in a mixture using gel electrophoresis. Understanding the changes in the expression of one or more of these proteins is critical to evaluating barrier integrity and permeability in health and disease. Furthermore, studying them will provide insight into the associated downstream signaling pathways and evaluation of therapeutic approaches for regulating BBB permeability. Herein, we have described the protocol for immunoblot analysis of ZO-1 as an indicator of tight junction integrity in brain microvascular endothelial cells.

1. Introduction

Blood-brain barrier (BBB) is a semipermeable membrane separating systemic circulation from the brain parenchyma. Microvascular hyperpermeability, an abnormal extravasation of plasma proteins and fluid into extravascular space in brain occurs at the blood-brain barrier. Alterations in BBB integrity following inflammation leads to vascular hyperpermeability and vasogenic edema [1, 2]. Optimal functioning of the BBB is crucial for maintaining the homeostasis of the brain. Inter-endothelial tight junctions of the BBB are the key structural and functional elements that play a major role in maintaining BBB integrity [3]. Tight junction proteins include transmembrane occludin, claudins, junctional adhesion molecules and membrane bound tight junction proteins zonula occludes [ZO]. Zonula occludens-1 (ZO-1) are scaffolding molecules that link the transmembrane tight junctions and the actin cytoskeleton. ZO-1 plays an important role in maintaining the function of BBB including its permeability [3].

In vitro brain injury experimental studies have shown upregulation of IL-1β, a pro-inflammatory cytokine that can disrupt the BBB by deregulating the expression of tight junction proteins such as ZO-1 [4, 5]. ZO-1, a 220 kDa tight junction protein can be measured to determine its role in determining tight junction integrity, by immunoblotting (Figure 1). The current western blot protocol outlines a detailed technique on how the proteins are resolved over an SDS polyacrylamide gel and transferred to a nitrocellulose membrane to be detected by the ECL detection technique.

Figure-1.

Figure-1

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Western blot analysis of Zonula Occludens-1 (ZO-1) from rat brain microvascular endothelial cell (RBMEC) lysates. RBMECs were exposed to increasing concentrations of hydrogen peroxide (H2O2) followed by immunoblot analysis. H2O2 treatment showed no significant effect on ZO-1 protein expression at the concentrations tested (ZO-1 was normalized to β-actin expression). This data was taken from a previously published article from our lab (Anasooya Shaji et al., Scientific Reports, 2019, 9.133). Creative Commons License https://creativecommons.org/licenses/).

2. Materials

  1. Rat Brain Microvascular Endothelial Cells (RBMVECs) [Cell Applications Inc., San Diego, CA].

  2. RBMVEC Media [Cell Applications Inc., San Diego, CA].

  3. Fibronectin [Sigma Aldrich, St. Louis, MO].

  4. Interleukin-1β, human [Sigma Aldrich, St. Louis, MO].

  5. ZO-1 Monoclonal Antibody [Thermo Fisher Scientific, Carlsbad, CA].

  6. Opti-MEM (1X)/reduced serum medium [Thermo Fisher Scientific, Carlsbad, CA].

  7. HyClone Dulbecco’s phosphate-buffered saline (PBS, without calcium, magnesium, or phenol red) [Thermo Fisher Scientific, Carlsbad, CA].

  8. Anti-rabbit IgG-FITC secondary antibody [Santa Cruz Biotechnology, Inc. Santa Cruz, CA].

  9. NuPAGE Novex® 10% Bis-Tris protein gels [Thermo Fisher Scientific, Carlsbad, CA].

  10. NuPAGE® MOPS SDS Running Buffer [Thermo Fisher Scientific, Carlsbad, CA].

  11. RIPA cell lysis buffer [Thermo Fisher Scientific, Carlsbad, CA].

  12. NuPAGE® Transfer Buffer [Thermo Fisher Scientific, Carlsbad, CA].

  13. Nitrocellulose/Filter Paper Sandwich [Thermo Fisher Scientific, Carlsbad, CA].

  14. Protease Inhibitor Cocktail (100X) [Thermo Fisher Scientific, Carlsbad, CA].

  15. Pierce™ ECL Plus Western Blotting Substrate [Thermo Fisher Scientific, Carlsbad, CA].

  16. Goat anti-mouse IgG-HRP [Santa Cruz Biotechnology, Inc. Santa Cruz, CA].

  17. CL-XPosure™ Film [Thermo Fisher Scientific, Carlsbad, CA].

3. Methods

3.1. RBMEC Cell Lysate preparation

  1. Rat Brain Microvascular Endothelial Cells (RBMVECs) are cultured n a 10 cm petri dish coated with 5% fibronectin (50 μg/ml in PBS).

  2. Once cells reach 80% confluency, remove the culture media and incubate the cells in reduced serum medium.

  3. Wash cells twice with ice-cold PBS and incubate in 1mL of ice-cold RIPA cell lysis buffer (1X) along with protease inhibitor cocktail (1X) for 5 minutes.

  4. Use a cell scraper to dissociate the cells from the petri dish. Collect the soup into a 1.5ml microcentrifuge tube and sonicate for 30 seconds on ice to fragment the cells.

  5. Centrifuge the lysate at 14,000g for 10 minutes at 4°C.

  6. Carefully collect the supernatant in a fresh centrifuge tube and determine protein concentration using BCA protein assay kit.

3.2. Western blot analysis of ZO-1

  1. Prepare equal amounts of total protein (50 G) in loading buffer and add β -mercaptoethanol to a final concentration of 5% (v/v%), heat at 95°C for 5 minutes on a heating block and transfer onto the ice, and prepare SDS-PAGE setup.

  2. Load protein ladder (5μl).

  3. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% Bis-Tris precast gels at constant voltage (120 V) for 90 minutes in MOPS SDS running buffer.

  4. Open the gel plates using a spatula, and immerse the gel in DI water to remove any traces of running buffer.

  5. Add 200 ml of methanol to 800 ml of 1X NuPAGE® Transfer Buffer to prepare protein transfer buffer.

  6. Soak nitrocellulose membrane and filter paper in transfer buffer to equilibrate, transfer the SDS-gel on to the filter paper and gently place the nitrocellulose membrane on top followed by a second filter paper on top of it.

  7. Create a sandwich by placing the filter – nitrocellulose – gel – filter paper between two blotting sponge pads and transfer it into blotting cassette. Insert the blotting cassette into protein transfer setup with the proper orientation.

  8. Proteins are transferred onto the nitrocellulose membrane at constant voltage (30 V) overnight.

  9. Following day remove the membrane from the transfer setup and block with 5% nonfat dry milk in Tris-Buffered Saline (TBS) with 0.05% Tween-20 (Blocking buffer) for 1 hour.

  10. Incubate the membrane with primary mouse monoclonal anti ZO-1 antibody diluted at 1:250 in 5% nonfat dry milk TBS-T overnight at 4°C.

  11. Next day wash the membrane three times with TBS-T.

  12. Block the membrane in 5% nonfat dry milk in Tris-Buffered Saline (TBS) with 0.05% Tween-20 for 1 hour at room temperature.

  13. Incubate the membrane with the goat anti-mouse IgG-HRP conjugated secondary antibody diluted at 1:2000 in 5% nonfat dry milk in Tris-Buffered Saline (TBS) with 0.05% Tween-20 for 2 hours at room temperature.

  14. Wash the membrane in TBS-T with wash buffer replaced every 15 minutes for a total of three washes.

  15. Prepare fresh ECL western blotting substrate and incubate the membrane in the substrate for 1 minute at room temperature.

  16. Pick the membrane and dab the edges to remove excess solution, cover it in a plastic wrap, use a roller to remove any trapped bubbles before placing in the film cassette.

  17. Expose the membrane to an x-ray film and develop using an autoradiogram.

  18. Developed x-ray films are scanned and the band intensity is quantified using ImageJ software.

4. Notes

  1. During the protein extraction process from the cells, the sonication step produces excess heat which can affect the proteins and so the process must be done on ice.

  2. Fill empty wells with loading buffer so that all the wells run equally and make sure to use the right molecular weight protein ladder that is compatible with the gel and the running buffer.

  3. While running the acrylamide gel running buffer gets hot overtime at high voltages, do not run the gel for too long at high voltage (140V+) as this will damage the gel.

  4. Since the transfer buffer contains 20% methanol do not use old buffer always prepare fresh transfer buffer. Old transfer buffer can be used for initial steps where the nitrocellulose membrane and the filter papers need to be soaked before setting up the transfer cassette.

  5. Protein transfer efficiency onto nitrocellulose membrane from the acrylamide gel is better when the transfer buffer is maintained at or close to 4°C, use either ice packs or run the transfer in a 4°C refrigerator overnight at low voltage.

  6. The membrane should not dry during any of the primary and secondary antibody incubation, washing steps.

  7. All membrane washes in TBS-T and the secondary antibody incubation should be done at room temperature.

  8. ECL western blot substrate should be used within one hour of preparation. The substrate should be prepared right before the final membrane wash post-secondary antibody incubation.

  9. Before the membrane is placed in the cassette all the trapped bubbles on the membrane should be removed using a roller to avoid poor signals during exposure.

  10. The membrane should be exposed to the x-ray film in the dark, and the exposure time can be adjusted based on the signal intensity.

  11. Sample loading can be verified by assessing β-actin protein expression.

  12. Use untreated cells to serve as controls.

ACKNOWLEDGMENTS

The authors acknowledge the support from National Institutes of Health (NIH) grant 5 SC3 NS127765-02 (BT)

References

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