Mouse anti sox2

Twenty-four hours after incubation with the inhibitors, P3 hypothalamic neurospheres were plated in Poly-D-Lysine coated 12-well culture plates and kept in culture for 1 day with fresh medium (DMEM-F12/Glutamax supplemented with growth factors) before fixation.
P3 hypothalamic neurospheres and differentiated hypothalamic neuroprogenitor cultures were fixed with 4% paraformaldehyde (Sigma) and permeabilized with 1% Triton X-100 (Sigma) followed by one hour blocking with 3% BSA (Sigma) and incubation with the primary antibody overnight at 4°C. Thereafter, the incubation with the secondary antibody was performed for one hour at room temperature. Nuclei were stained with 4′6-diamidino-2-phenylindoline, DAPI (1∶5000, Applichem).The primary antibodies used were: mouse anti-Ki-67 (1∶50; NovoCastra), mouse anti-Neu-N (1∶500, Chemicon) and mouse anti-SOX-2 (1∶200, R&D Systems). The secondary antibodies used were: goat anti-mouse Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 (1∶200, Invitrogen).

For Western blot, cells were lysed in cold radioimmunoprecipitation buffer (50 mM Tris–HCl pH 7.5, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors, centrifuged at 14,000 rpm for 20 min at 4 °C, and supernatant was collected as whole cell extract. 40 μg of proteins were resolved by SDS-polyacrylamide electrophoresis in 10% to 12% gels and transferred by electroblotting to a nitrocellulose membrane.
For protein immunoprecipitation and coimmunoprecipitation experiments, cells were lysed as already reported (46 (link)). Briefly, 500 μg whole cell extract was incubated overnight with 50 μl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, #sc-2003) and 2.5 μg of mouse anti-SOX2 (R&D System, #MAB2018), mouse anti-p38 (Santa Cruz Biotechnology, #81621), or normal mouse IgG (Santa Cruz Biotechnology, #sc-2025). Proteins were eluted after boiling for 10 min at 95 °C in 2× Laemmli sample buffer (Bio-Rad, catalog no.: 1610747) and detected by using SuperSignal West Femto (ThermoFisher Scientific) and imaged with ChemiDocTM Imaging Systems (Bio-Rad). A list of primary antibodies used is reported in Table S5.

For pluripotency marker stainings, iPSC colonies were passaged as described above and grown on Matrigel^TM-coated coverslips in ES medium containing, 50% MEF-conditioned media (own preparation) supplemented with 5 ng/ml FGF2 (Sigma). Colonies were then stained for alkaline phosphatase according to the manufacturer’s protocol (Millipore) or were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT for analysis of pluripotency markers by immunofluorescence. Fixed colonies were incubated for 2 h in blocking solution (3% normal horse serum and 0.05–0.2% Triton-X100 in PBS). Plates were incubated over night at 4 °C using the following primary antibodies: rabbit anti-Nanog (1:500), rabbit anti-Oct4 (1:1000), mouse anti-SSEA4 (1:500), mouse anti TRA-1-60 (1:500) (all from Abcam) and mouse anti-Sox2 (1:500, R&D Systems). Differentiated EBs were stained with rabbit anti-α-SMA (1:500, Sigma Aldrich), mouse anti-α-Fetoprotein (1:500, Abcam), rabbit anti-GATA4 (1:500, Abcam), and mouse anti-TUJ1 (1:1000, Covance), mouse anti-Actinin (Sigma, 1:200) and mouse anti Beta-Catenin (BD Bioscience 1:500).

Aliquots of protein (30 µg) were subjected to Western blotting. The membranes were incubated with a rabbit polyclonal EGFR antibody (Santa Cruz Biotechnology, USA), a mouse anti-vimentin (Santa Cruz Biotechnology, USA), a mouse anti-NANOG (Santa Cruz Biotechnology, USA), a mouse anti-Sox2 (R&D Systems, USA), a rabbit anti-N-cadherin (Abcam, USA), a rabbit anti-fibronectin 1 (Merck, USA), a mouse anti-CD44 (Cell Signaling, USA) and anti-E-cadherin (Santa Cruz Biotechnology, USA). After washing with TBS-Tween (Merck, USA), the membranes were incubated with HRP-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, USA) or HRP-conjugated goat-anti-mouse antibody (Santa Cruz Biotechnology, USA). Equal loading was verified by reprobing the membranes with HRP-conjugated anti-GAPDH antibody (Novus Biologicals, USA). The bands were visualized with Western Blotting Luminol Reagent (Bio Rad, USA) using Chemi Doc™ MP Imaging System (Bio Rad, USA).