Anti p75

The NLBs obtained from the different conditions of culture were enzymatically and mechanically dissociated to obtain a single-cell suspension in which we carried out the cell labeling. The cells were fixed with 4% (weight/volume) paraformaldehyde in 0.1 mol/l phosphate-buffered saline. Then, they were incubated for 1 hour in 2.5% (weight/volume) bovine serum albumin in phosphate-buffered saline and with primary (16 hours at 4°C) and secondary antibodies (1 hour at room temperature). Several primary antibodies were used: anti-Nestin (goat polyclonal; 1:1000) (Santa Cruz Biotechnology, Inc) as a neural precursor cells marker, anti-β III Tubulin (mouse monoclonal; 1:500) as a neuronal precursor cells marker (Merck Millipore, German) and anti-p75 (rabbit polyclonal; 1:500) as a enteric precursor cells marker (Abcam, UK). The secondary antibodies were anti-goat IgG labeled with Cy5, anti-mouse Ig G labeled with Cy2 (1:200; Jackson Immuno Research Laboratories Inc, UK), and anti-rabbit IgG labeled with Alexa Fluor 568 (1:200; Life Technologies, USA) respectively. Flow cytometry analysis data were collected using the LRS II Fortessa cell analyzer (Becton Dickinson, USA). The expressions of Nestin, βIII-Tubulin and p75 markers as well as of different combinations of these markers were analyzed in the EPCs.

Control and treated cells were collected and lysated as previously described and the protein amount were evaluated [43 (link), 44 (link)]. 30 μg of proteins were loaded and separated on precast 4–20% gradient Bis-Tris gel in running buffer at 100 mV for 70 min followed by transfer to PVDF membranes using a semi-dry device (Thermo scientific, UK), then blocked in 5% no-fat milk for 30 minutes. Membranes were incubated with the subsequent primary antibodies overnight: anti-p-AKT (1:1000), anti-PI3K (1:1000 Cell Signaling, USA), anti p-CREB (1:500 Cell Signaling, USA), anti-pTrkB (1:2000 Cell Signaling, USA), anti-mBDNF (1:500 Abcam, UK), anti-pJNK (1:1000 Santa Cruz, USA), anti-p75(1:1000 Abcam, UK), anti-pro-BDNF(1:1000 Millipore, USA), anti-pERK5 (1:1000 Cell Signaling, USA), anti p-ERK1,2 (1:1000 Santa Cruz, USA). After different washes, membranes were incubated with 1:10000 horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein bands were detected, normalized and analyzed to actin (housekeeping). Anti-β-actin (HRP-conjugate) (1:10000) has been used. To reprobe, membranes have been stripped with Restore stripping buffer (Thermo Scientific, UK) following manufacturer’s instructions.