β mercaptoethanol

All mESC lines were grown without feeder cells on gelatin-coated flasks (Millipore, 0.1%) in serum-containing ES cell medium (DMEM (Sigma), 15% FBS (PanBiotech), 0.1 mM β-Mercaptoethanol (Sigma), 1000 U/ml leukemia inhibitory factor (LIF, Merck)). mESCs were passaged every second day at a density of 4 × 10⁴ cells/cm² and medium was changed daily. Cells were differentiated by LIF withdrawal in DMEM supplemented with 10% FBS and 0.1 mM β-Mercaptoethanol at a density of 2 × 10⁴ cells/cm² on fibronectin-coated dishes (Merck, 10 μg/ml).
For the differentiation of mutant cell lines (Fig. 4g), cells were first adapted to 2i + LIF medium (ES cell medium with addition of 3 μM Gsk3 inhibitor CT-99021 (Axon Medchem) and 1 μM Mek inhibitor PD0325901 (Axon Medchem)) for at least five passages before undergoing differentiation via LIF withdrawal (see above). TX1072 XX and XO cells were grown in ES cell medium supplemented with 2i and differentiated by 2i/LIF withdrawal. Hek293T cells were cultured in DMEM supplemented with 10% FBS and passaged every 2 to 3 days.

The D1 cell line is a growth factor-dependent spleen-derived immature DC cell line from C57BL/6 (H-2^b) mice. D1 cells were cultured in IMDM medium supplemented with GM-CSF supernatant^{50 (link)}. The B3Z, OTIIZ, and 3A12 hybridoma cell lines were cultured in IMDM medium (Lonza, Basel, Switzerland) supplemented with 8% FCS (Greiner, Kremsmünster, Austria), penicillin and streptomycin, glutamine (Gibco, Carlsbad, CA, USA), β-mercaptoethanol (Merck, Kenilworth, NJ, USA), and hygromycin B (AG Scientific Inc., San Diego, CA, USA) to maintain expression of the beta-galactosidase reporter gene. The B16OVA and TC-1 tumor cell lines were cultured in IMDM medium (Lonza, Basel, Switzerland) supplemented with 8% FCS (Greiner, Kremsmünster, Austria), non-essential amino acids, sodium pyruvate, glutamine, penicillin and streptomycin, (all from Gibco, Carlsbad, CA, USA), β-mercaptoethanol (Merck, Kenilworth, NJ, USA). G418 (Life Technologies, Carlsbad, CA, USA) was used to maintain OVA expression in B16OVA cells and E6 and E7 expression in TC-1.

Human hair was obtained from a local hair salon with a customer permission and thoroughly washed. External lipids were removed using a mixture of chloroform and methanol (2:1, v/v) for 24 h. After removal of the solvents, the hair was dried in a chemical hood. 1 g of delipidized hair was mixed with 20 mL of Shindai solution (sol-ME) [14 (link)] containing 25 mM Tris.HCl, pH 8.5 (Merck, Kenilworth, NJ, USA), 2.6 M thiourea (Sigma-Aldrich, St. Louis, MO, USA), 5 M urea (Sigma-Aldrich, St. Louis, MO, USA) and 0.717 M β-mercaptoethanol (Merck, Kenilworth, NJ, USA) or of a modified Shindai solution (sol-DTT) containing 0.102 M 1,4-dithiothreitol (Merck, Kenilworth, NJ, USA) instead of β-mercaptoethanol. Extraction was performed for 72 h under shaking at 50 °C. Proteins were also extracted from human nails and bovine hooves using sol-DTT as described above. After extraction, the protein solution was filtered using medical gauze, dialyzed and centrifuged at 12,000× g for 30 min at 20 °C. The concentration of protein released from each sample was monitored by the colorimetric Bradford method [32 (link)] using the Bio-Rad protein assay reagent [33 ] and bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as a standard.