Ep2000y

To localize the γ-secretase complex in sympathetic neurons, neuronal cell cultures were treated in the presence or absence of BMP-7 (50 ng/mL) for 5 d, fixed with 4% paraformaldehyde and immunostained for presenilin 1 (PSEN1), presenilin 2 (PSEN2), nicastrin, PEN2 or APH1a, and visualized using indirect immunofluorescence. The following primary antibodies were used: rabbit anti-PSEN1 (EP2000Y, 1:500, Abcam, Cambridge, MA), rabbit anti-PSEN2 (ab51249, 1:100, Abcam, Cambridge, MA), rabbit anti-nicastrin (ab45425, 1:50, Abcam, Cambridge, MA), mouse anti-PEN2 (1C12-G5, 1:10, Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-APH1a (ab12104, 1:50, Abcam, Cambridge, MA). Alexa dye conjugated goat anti-rabbit (1:1000, Thermo Fisher Scientific, Waltham, MA) was used as the secondary antibody. The antibodies have been previously used to detect members of the γ-secretase complex (D' Souza et al., 2015; Wang et al., 2015; (link)Zhang et al., 2018) (link).
To detect phospho-SMAD 1,5, neurons were treated on day in vitro (DIV) 4-5 with BMP-7 (50 ng/mL) in the presence or absence of the γ-secretase inhibitors for 5 d. Subcellular distribution of SMAD1 was assessed in cultures immunostained for phosphorylated SMAD 1,5 (41D10, 1:100 dilution, Cell Signaling Technology) and visualized using indirect immunofluorescence as previously described (Kim et al., 2009) .