TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (3) Western Blot Analysis

doi:10.1038/nprot.2007.382

Method Article

TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (3) Western Blot Analysis

https://doi.org/10.1038/nprot.2007.382

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Genetics

Biological techniques

Molecular Biology

Biochemistry

Computational biology and bioinformatics

experimental autoimmune uveoretinitis (EAU)

IL-27p28 immunohistochemistry and confocal microscopy

intracellular cytokine staining assay

retinal organ culture

IL-27 is constitutively expressed in retina and inhibits T cells that mediate EAU

IL-27 is predominantly expressed in antigen-presenting lymphoid cells such as dendritic cells. Using a combination of RT-PCR, Western blot analyses we demonstrate expression of IL-27 in the retina and retina cells. Expression of IL-27 was localized to the retinal ganglion and photoreceptor cells as well as the retina pigmented epithelium (RPE) by immunohistochemical analysis. Expression of IL27 in the retina was found to be positively regulated in retinal cells by the proinflammatory cytokine, IFN-γ and chromosome immunoprecipitation assay (Chip) revealed direct binding of STAT1 transcription factor to the IL27 promoter.

RT-PCR and Quantitative RT-PCR (qRT-PCR)

All RNA samples were DNA-free and cDNA was generated with SuperScript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) and oligo(dT)12–16.

Perform RT-PCR with AmpliTaq Gold DNA polymerase (ABI, Foster City, CA) as described ⁷. (For genes with introns, each gene-specific primer pair was designed to span at least an intron. All cDNA preparations used were found to be suitable substrates for PCR amplification on the basis of efficient amplification of a β-actin sequence.)

Real-time RT-PCR (qRT-PCR) analysis was performed on an ABI 7500 (Applied Biosystems, Foster City, CA) as previously described ⁸ using primers and probes obtained from Applied Biosystems. The mRNA expression levels were normalized to both β-Actin and GAPDH house keeping genes. (see above for step-by-step procedures)

Western blot analyses

ARPE-19 or human Muller cell line (MIO-M1 were starved for 2 hours and cultured in DMEM supplemented with IFNγ for 15, 30, 60, 90 minutes in the presence or absence of AG-490 or the protein synthesis inhibitor, cycloheximide. Blots were probed with rabbit polyclonal IL-27 antibody or pSTAT1, IRF-1, IRF-8, β-Actin specific antibodies. Pre-immune serum was used in parallel as controls and signals were detected with the ECL-PLUS system.

The steps described in this protocol are:

Stimulation of cultured cells with Cytokines and preparation of whole cell extracts

Electrophoresis

Semi-Dry Transfer Assembly

Hybridization

Detection with ECL-plus

Other protocols related to our Nature Medicine paper can be found here:

"Detection of TH17 cells in human blood":http://www.natureprotocols.com/2007/08/30/th17_cells_contribute_to_uveit.php

"Analysis by confocal microscopy":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_3.php

"Chromatin immunoprecipitation":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_4.php

"Retinal cell isolation":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_5.php

• DMEM containing L-glutamax I (GibcoBRL).

• ARPE-19, was obtained from American Type Culture Collection (Manassas, VA, USA)

• Human recombinant IFN-γ (Invitrogen, Gaithersburg, MD)

• Cycloheximide (CHX) (Sigma Chemical Co., St. Louis, Mo)

• Streptomycin (Sigma)

• AG-490 (Calbiochem, San Diego, CA)

• Penicillin/Streptomycin (Invitrogen).

• Amphotericin B (Invitrogen).

• Rabbit polyclonal IL-27 antibody (Cat# IMG-5096A; Imgenex, San Diego, CA)

• pSTAT1, IRF-1, IRF-8, β-Actin specific antibodies (Santa Cruz or Invitrogen ).

• ECL-PLUS system (Amersham, Arlington Heights, IL)

• RIPA (500 ml)

490 ml 1xPBS

5 ml NP40 (1%)

2.5g Sodium Deoxycholate

5 ml SDS (use 10% stock)

• Stock Solutions

100 mM NaV04 (Sodium Vanadate)

1 M NaF (Sodium Flouride)

1 M Benzamidine (can omit)

5 M NaCl

PMSF (dissolve 50 mg in 5 ml isopropanol): stable at 25ºC for 9 months

• Protease Inhibitor Cocktail Set I (Calbiochem cat# 539131)

100x Stock (dissolve content of bottle in 1 ml water)

Store in 0.5 ml aliquots

• Lysis Buffer

RIPA 46 ml

Complete Protease inhibitor (Roche) 1 tablet

100x Protease Inhibitor Cocktail Set I 0.5 ml

NaF 2.5 ml (50 mM)

NaV04 0.5 ml (1 mM)

Benzamidine (can substitute with RIPA) 0.5 ml (10 mM)

5M NaCl 1.5 ml (150 mM)

Reagent Setup

Cells and culture conditions

Human Müller cell line (MI0-M1) was provided by G. Astrid Limb (Institute of Ophthalmology, London, UK) and originated from spontaneously immortalized Müller cells from an eye of a 68-year-old female corneal donor. MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) cells were cultured in DMEM containing L-glutamax I. The human retina pigmented epithelial cell line, ARPE-19, was cultured in minimum essential medium (MEM). Both cell lines were propagated in medium supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were rinsed twice in serum-free media (SFM) and incubated in SFM containing 100 U/ml human recombinant IFN-γ or AG-490 (100 μM) for 30 min or 90 min at 37 °C, 5% CO2. Protein synthesis inhibitor, cycloheximide (CHX) was used at 35 µg/ml and was added 30 minutes before addition of IFN-γ. CHX treatment allows for discrimination between effects of IFN-γ that require de novo protein synthesis and those that do not.

Stimulation with Cytokines and preparation of whole cell extracts

1| Grow cells as indicated above upto 80% confluent.

2| Replace medium with starvation medium (1% BSA) and starve for 2 h

3| Stimulate starved cells with cytokines or growth factors as described avove.

4| After stimulation, rinse cells with ice-cold PBS.

5| Following steps should be done on ice or at 4 °C using ice cold buffers.

6| Add 0.6 ml of lysis buffer (with freshly added inhibitors) to a 100 mm cell culture plate.

7| Scrape plate with a cell scraper.

8| Using 2 ml pipette or syringe fitted with 21 gauge needle, transfer lysate to chilled microcentrifuge tube.

9| Wash the plate once with 0.3 ml of lysis buffer and combine with first lysate.

10| Pass through the 21 gauge needle to shear the DNA.

11| Add fresh 10 µl stock PMSF (10 mg/ml) and incubate on ice for 30 minutes.

12| Microcentrifuge cell lysate (Max speed in microfuge) for 30 minutes at 4° C.

13| Collect supernatant in a fresh tube

14| Centrifuge supernatant again for 10 minutes. The supernatant fluid is the total cell lysate.

15| Aliquot sample for freeze at -70°C

16| Quantitate protein extract by photometry

Electrophoresis

1| Use approximately 20-50 μg of purified protein, nuclear or whole cell extract

2| Mix with 4X Nupage sample buffer (i.e make it 1X with respect to buffer).

3| Denature proteins (10 min at 70 degrees) in loading buffer with reducing reagent and vortex to mix

4| Wash wells and load 10-20μl of sample.

5| Run at 200 V until the first blue band is at the bottom (35-50 min).

6| Semi-Dry Transfer Using Immobilin-P Membrane

7| Soak gel in transfer buffer for 15 minutes (with 3 buffer changes).

8| Wet membrane in 100% methanol for 15 seconds

9| Wet membrane in H₂O for 2 minutes

10| Wet membrane in transfer buffer for at least 5 minutes

Semi-Dry Transfer Assembly

1| Wet two sheet of thick filter paper in transfer buffer

2| Place on semi-dry blotter.

3| place the treated membrane on top of buffered filter papers

4| place the gel on top of the membrane

5| Wet two thick filter papers in transfer buffer and place on top of the gel

6| Run at 16-20 V (200 mAmp), for approx. 30 min. Don’t exceed 25 V

Hybridization

1| Rinse membrane in water (2X)

2| Incubate 1 hr with 5% MLK/0.05% Tween 20/PBS (MTB) without Mg/Ca++ at RT

3| Incubate with primary Ab in MTB (0.1-1 μg per ml) at 4 °C overnight

4| Rinse in water (2X)

5| Incubate with anti-rabbit or anti-mouse HRP (1: 10,000-25,000 dilution) at RT for 1 hour

6| Rinse with PBS+0.05% Tween 20 twice and wash for 10 min at RT (3 times).

Detection with ECL-plus

1| Remove detection reagents and allow to equilibrate at room temperature before opening

2| Mix detection solutions A and solution B in a ratio of 40:1(Total volume 0.1ml/cm²)

3| Pipette detection solution on the membrane and incubate for 5 min at room temperature.

4| Place on Saran Wrap

5| Autoradiograph.

Nussenblatt, R.B. Proctor Lecture. Experimental autoimmune uveitis: mechanisms of disease and clinical therapeutic indications. Invest Ophthalmol Vis Sci 32, 3131-3141 (1991).
Gregerson, D.S., Obritsch, W.F., Fling, S.P. & Cameron, J.D. S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis. Journal of immunology (Baltimore, Md.) 136, 2875-2882 (1986).
Wacker, W.B., Donoso, L.A., Kalsow, C.M., Yankeelov, J.A., Jr. & Organisciak, D.T. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. Journal of immunology (Baltimore, Md.) 119, 1949-1958 (1977).
Egwuagu, C.E., Mahdi, R.M., Nussenblatt, R.B., Gery, I. & Caspi, R.R. Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens. J Immunol 151, 1627-1636. (1993).
Pepperberg, D.R., Okajima, T.L., Ripps, H., Chader, G.J. & Wiggert, B. Functional properties of interphotoreceptor retinoid-binding protein. Photochem Photobiol 54, 1057-1060 (1991).
Takase, H., et al. Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins. J Neuroimmunol 168, 118-127 (2005).
Yu, C.R., et al. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J Biol Chem 278, 29752-29759 (2003).
Egwuagu, C.E., et al. Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance. J Immunol 168, 3181-3187. (2002).

This research was funded by the Intramural Research Programs of the NEI and NIH. Authors thank Dr. Wei Zhu (Laboratory of Immunology, NEI, NIH) for assistance with fundoscopy.

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TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (3) Western Blot Analysis

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TH17 cells contribute to uveitis and scleritis and are inhibited by IL-27/STAT1 in the retina. (3) Western Blot Analysis

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Introduction

Reagents

Procedure

References

Acknowledgements

Associated Publications

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