β mercaptoethanol

E14 mES cells were cultured under feeder-free conditions on 0.5% gelatin-coated dishes with mouse embryonic stem cell (ESC) medium consisting of knockout DMEM (Gibco), 10% FBS (Gibco), 2 mM L-glutamine (Merck Millipore), 1% nonessential amino acids (Merck Millipore), 0.3 mM β-mercaptoethanol (Nacalai Tesque), and 1000 U/ml LIF (Merck Millipore) at 37 °C and 5% CO₂. For the differentiation assay, mES cells were cultured in mouse ESC medium without LIF and supplemented with 0.5 μM retinoic acid25 (link). To change from serum + LIF to 2i + LIF conditions, E14 mES cells cultured with serum + LIF were subcultured in 2i + LIF medium (N2B27 medium [Life Technologies] with 1 μM PD0325901 and 3 μM CHIR99021 [Stemgent], together known as 2i medium, and 1000 U/ml LIF). After several passages, the adapted cells were used for study. For the differentiation assay under 2i + LIF conditions, mES cells were cultured without 2i + LIF20 (link)21 (link).

Bone marrow (BM) cells freshly isolated from C57BL/6 mice were cultured in RPMI 1640 medium (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) supplemented with 10% FBS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 10 mmol/L, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher Scientific, Waltham, MA, USA), 1% minimum essential medium non-essential amino acids (MEM NEAA) 10 mL/L, (Thermo Fisher Scientific), 1 mmol/L sodium pyruvate (Thermo Fisher Scientific), 50 nmol/L β-mercaptoethanol (Nakalai Tesque, INC, Kyoto Japan), penicillin-streptomycin-glutamine (100 U/mL penicillin, 100 µg/mL streptomycin and 29.2 mg/mL glutamine) (Thermo Fisher Scientific, Waltham, MA, USA), in the presence of GM-CSF (10 ng/mL) (PeproTech, Cranbury, NJ, USA). Medium was refreshed and GM-CSF was added, twice in 8 days. On day 10, non-adherent cells were harvested. These cells were purified immunomagnetically by two or three rounds of positive selection with CD11c (N418) MicroBeads (Miltenyi Biotec B.V. & Co., KG, Bergisch Gladbach, Germany) [41 (link)]. Purified BMDCs were seeded in 12-well or 24-well dishes, pre-treated with or without U0126 for 1h, and then subsequently treated with or without IL-4 (Peprotech) for 24 h. Culture supernatants were collected at 24 h and analyzed by ELISA. Cells were also collected for PCR analysis and western blot analysis as described below.

For the multitissue expression analyses, brain, thymus, lung, heart, liver, spleen, kidney, testis, epididymis (caput, corpus, and cauda regions), seminal vesicle, prostate (mixture of dorsal, lateral, and ventral regions), coagulating gland (also known as anterior prostate), ovary, and uterus were collected from adult C57BL/6J mice. These samples were processed in TRIzol (Ambion). For Western blot analysis, TGC proteins were extracted with lysis buffer containing Triton-X 100 (50 mM NaCl, 10 mM Tris⋅HCl, 1% [vol/vol] Triton-X 100 [Sigma Aldrich], pH 7.5) containing 1% (vol/vol) protease inhibitor mixture (Nacalai Tesque). Proteins of cauda epididymal spermatozoa were extracted with sample buffer containing β-mercaptoethanol (Nacalai Tesque) as described previously (38 (link)).

Whole cell lysates from 5.0 × 10⁶ cells, prepared using an SDS-based buffer (5 mM EDTA, 1% SDS) supplemented with Protease inhibitor cocktail (Roche) and PhosSTOP EASY (Roche), were mixed with an equal volume of twofold concentrated sample buffer (Bio-Rad Laboratories) containing β-mercaptoethanol (Nacalai Tesque), and were treated for 5 min at 100°C. Immunoblot analysis was performed as described previously using a mouse anti-FLAG antibody (Millipore, clone JBW301) or a mouse anti-α-tubulin monoclonal antibody (AA13, Funakoshi).