Ab24741

The following antibodies were used for labeling: affinity-purified rabbit polyclonal anti-APH-1a (B80.3) and affinity-purified monoclonal anti-NCT-CT 9C3 (Annaert et al., 2001 (link); Esselens et al., 2004 (link)); affinity-purified rabbit polyclonal against N-terminal amino acids 103–124 of NCT (anti-NCT-ECD, ab24741) and affinity-purified rabbit polyclonal against 13 amino acids at the C-terminus of PEN-2 (anti-PEN-2, ab62514) (both from Abcam); rabbit polyclonal against C-terminal amino acids 450–467 of PS-1 (anti-PS1-CT, S182; Sigma). The following antibodies were used for detection by western blotting: rabbit anti-PEN-2 (B126.1; Annaert et al., 2001 (link); Esselens et al., 2004 (link)); anti-human-PS1-loop (MAB5232, Chemicon); mouse anti-NCT (BD Transduction Laboratories); rat anti-PS1-NTF (MAB1563, Millipore); and anti-FLAG M2 (Sigma).

Cells were lysed in pre-cooled RIPA cell buffer (P0013B, Beyotime Biotechnology, China) containing protease inhibitor (DI111-02, TRANSGEN BIOTECH, China, 1: 100). Proteins were separated by a 10% SDS-PAGE gel at RT and electrotransferred to a PVDF membrane in ice-bath. Probing was performed with specific primary antibodies and HRP-conjugated secondary antibodies. The primary antibodies used were PIAS1 (ab2474-1, Abcam, 1:1000) and GAPDH (SC-8035, Santa Cruz, 1:2000). Proteins were detected in super ECL detection reagent (No.180-501, Tanon, China).