Mouse anti sox17

For detection of stage-specific markers, cells were grown and differentiated in 96 well plates (μClear, Greiner). Cells were rinsed twice with PBS, fixed in 4% paraformaldehyde (USB) for 15 min at room temperature (RT) and then washed twice with PBS and blocked for 30 min at RT in 1% BSA (Life Technologies) and 0.1 mg/ml human IgG (Sigma) in perm/wash buffer (BD). Cells were subsequently stained for 2 hours at RT or overnight at 4°C with rabbit anti-OCT4 (Cell Signalling), mouse anti-SOX17 (Abcam) or isotype control antibodies diluted in perm/wash buffer. Cells were washed with perm/wash buffer and incubated at 4°C in the dark with goat anti-mouse-FITC and chicken anti-rabbit-Cy5 (Molecular probes) diluted 1:400 in perm/wash buffer. After 1h of incubation, cells were washed with PBS and incubated with Hoechst 33342 (Life Technologies) for 15 min at room temperature. After final washing with PBS 96 well plates were then imaged with IN Cell Analyzer 2000 (GE Healthcare).

Western blot analysis was performed as previously described.^{52 (link)} Blots were probed with 1/1000 rabbit anti-Oct4 (#2788), 1/1000 rabbit anti-MAP2 (#4542) (Cell Signaling Technology, Beverly, MA, USA), 1/500 rabbit anti-Foxj3 (#SAB2100844), 1/500 rabbit anti-Zbtb18 (#SAB2103436), 1/1000 mouse anti-Nestin (#MAB5326), 1/1000 rabbit anti-Brachyury (#B8436), 1/2000 mouse anti-β-actin (#A5441) (Sigma-Aldrich), 1/1000 goat anti-Flk1 (VEGF receptor 2, #AF644; R&D Systems, Minneapolis, MN, USA), 1/1000 mouse anti-Sox17 (#ab192453), 1/1000 rabbit anti-Foxa2 (#ab108422) (Abcam, Cambridge, MA, USA), and 1/1000 mouse anti-Tuj1 (neuronal class III β-tubulin, #MMS-435P; Covance, Princeton, NJ, USA). Immunoblots were revealed by autograph using SuperSignal west pico substrate (Thermo Fisher Scientific). The relative intensity of the protein bands was quantified using Image J software (NIH, Bethesda, MD, USA) and calculated by samples normalized to the controls. All data were presented as mean±S.D. and derived from three independent experiments.

Western blot analysis to assess protein expression was performed as previously described [30 (link)]. Primary antibodies included mouse anti-SOX17 (Abcam, Cambridge, UK) and mouse anti-GAPDH (Sigma, ST Louis MO, USA). All the other antibodies were purchased from CST (Boston, USA).