Anti nurr1

Interaction between Nurr1 and Foxa2 (in mouse VM tissue at 10 weeks of age) was assessed by IP. Tissues were lysed in IP lysis buffer (Thermo Scientific, Waltham, MA) supplemented with protease inhibitors. Lysates were incubated with anti-Nurr1 (1:1,000, mouse, R&D Systems) or anti-Foxa2 (1:1,000, goat, Santa Cruz) for 18–24 h at 4°C. The mixtures were shaken with magnetic beads (Life Technologies) for 1–2 h at room temperature. After washing, immunoprecipitated proteins were eluted in sample buffer and subjected to Western blot analysis with anti-Foxa2 (1:1,000, goat, Cell Signaling) or anti-Nurr1 (1:500, mouse, R&D Systems).

For immunofluorescence analysis, cells were fixed in 4% (Vol/Vol) paraformaldehyde for 15 min then blocked in 8% (Vol/Vol) HyClone Fetal Bovine Serum (Thermo Scientific) for 1 h at room temperature. This was followed by incubation with primary antibodies in blocking buffer containing 0.2% Saponin (Sigma-Aldrich) over night at 4°C. Cells were then washed with PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594, Invitrogen) at 1:500 for 1 h at room temperature. Cells were mounted in Vectashield mounting medium containing DAPI (VectorLabs) to label the nuclei. The following primary antibodies were used (all at 1:200 dilution unless otherwise stated): anti-LMX1 (Millipore), anti-Nurr1 (R&D Systems), anti-VMAT2 (Proteintech, 20873-1-AP), anti-GIRK2 (Proteintech, 21647-1-AP and Millipore, AB5200), anti-MAP2 (Millipore, MAB3418), anti-FoxA2 (Millipore, AB4125), anti-mTOR (Cell Signaling, 2972) 1:100, anti-phospho-mTOR-Ser2448 (Cell Signaling, 5536) 1:100, anti-S6 Ribosomal Protein (Cell Signaling, 2217) 1: 200, anti-phosphoS6-Ser235/236 (Cell Signaling, 2211 and 4856) anti-LAMP1 (U. Iowa Developmental Hybridoma Bank, H4A3) 1:100, anti-Tuj1 (Neuromics, MO15013 and BioLegend, 801207), anti-TFEB (Bethyl Laboratories, A303-673A), anti-Tyrosine Hydroxylase (Sigma-Aldirch, T1299), and anti-Bip/Grp78 (abcam, ab21685).