*4.7. Western Blot*

Approximately 300,000 LNSCs were collected in 50 μL of radioimmunoprecipitation assay (RIPA) buffer (Bioke, Leiden, The Netherlands) supplemented with leupeptin (1 μL/mL, Sigma Aldrich, Zwijndrecht, The Netherlands), pepstatin A (1 μL/mL, Sigma Aldrich, Zwijndrecht, The Netherlands), and PMSF (phenylmethanesulfonyl fluoride, 4 μL/mL, Sigma Aldrich, Zwijndrecht, The Netherlands) and were stored at −80 ◦C till further use. After thawing, protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific, Landsmeer, The Netherlands). Then, 10 μg of each sample, diluted in NuPAGE reducing agent and NuPAGE LDC buffer (Thermo Fisher Scientific, Landsmeer, The Netherlands) was loaded into a NuPAGE 4–12% gradient gel together with a Protein Molecular Weight Marker (Odyssey® One-Color, LI-COR, Bad Homburg, Germany). Gel was run in MES buffer (Thermo Fisher Scientific, Landsmeer, The Netherlands) for 1 h at 200 V. Subsequently, proteins were transferred onto a PVDF membrane (Immobilon-FL, Merck Millipore, Amsterdam, The Netherlands) for 70 min at 30 V. For washing TBST buffer (50 mM Tris, 150 nM NaCl, 0.1% Tween 20) was used and after blocking of membrane with 5% Blotting-Grade Blocker (Bio-Rad, Veenendaal, The Netherlands), the membrane was incubated with monoclonal mouse IgG1 anti human AIRE (1:500) or polyclonal goat IgG anti human Actin (1:2000) (both Santa Cruz, Huissen, The Netherlands) overnight at 4 ◦C. The next day the membrane was washed and incubated with anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) (both 1:2000, both Dako, Stockholm, Sweden) and were visualized using the HRP substrate Lumi-Light Plus (Roche, Woerden, The Netherlands). Blot was analyzed using ImageQuant LAS4000 (GE Healthcare, Zeist, The Netherlands).
