Skip to main content
. 2021 Feb 3;296:100372. doi: 10.1016/j.jbc.2021.100372

Figure 8.

Figure 8

PSA-NCAM1 is cleaved by BACE1.A, HEK cells were transiently transfected with the indicated expression vectors (NCAM1, ST8SIA2, and BACE1) for 48 h. Representative immunoblot of cell lysates (Lysate) and conditioned media (CM) using anti-PSA-NCAM1 (2-2B), anti-Myc (2272), anti-turboGFP (OTI2H8), anti-NCAM1 (AF2408), anti-BACE1 (D10E5), and anti-GAPDH (MAB374) antibodies. ST8SIA2 ectopic expression induces an increase in PSA-NCAM1 levels. Note that the ectopic expression of BACE1 produces BACE1-specific NCAM1-βCTFs (∼38 and ∼45 kDa) in the cell lysates and released sNCAM1β in the CM. Also, secreted soluble PSA-NCAM1 levels were increased. This result indicates BACE1 cleaves PSA-NCAM1 as well as non-PSA-NCAM1 in HEK cells. n = 3. B–D, NCAM1 proteins (PSA-NCAM1 and non-PSA-NCAM1) from P10 hemibrain lysates were immunoprecipitated using anti-C-terminal NCAM1 antibody (5B8). Immunoprecipitants were incubated with or without sialidase (P0722) to remove sialic acid modification and were then incubated with or without human recombinant BACE1 (h-BACE1) (B). After treatment, samples were directly analyzed by western blot (WB) using anti-PSA-NCAM1 (2-2B) (C) or anti-N-terminal-NCAM1 (AF2408) (D) antibodies. As expected, PSA-NCAM1 was almost absent in samples incubated with sialidase (lanes 1 and 3). Immunoblot with anti-N-terminal-NCAM1 (AF2408) antibodies revealed the presence of an N-terminal NCAM1 fragment in samples incubated with h-BACE1 independently of sialylation status of NCAM1 (arrowheads). E and F, immunoprecipitated endogenous APP using anti-C-terminal APP antibody (C1/6.1) was incubated with human BACE1 (h-BACE1) as a positive control for the BACE1 enzymatic cleavage experiment. While C99 and C89 represent BACE1-mediated C-terminal APP fragments (APP-βCTFs), C83 is an α-secretase-mediated C-terminal APP fragment (APP-αCTFs). APP-CTFs are present as phosphorylated (pC99, pC89, and pC83) and nonphosphorylated (C99, C89, and C83) forms. h-BACE1 cleaves APP-FL and increased C99 and pC99 levels, concurrently decreased C89/pC89 and C83/pC83 levels. Bands around at 50 kDa (double open arrowheads) and 25 kDa (open arrowheads) correspond to the heavy and light chain of precipitated primary antibody (C1/6.1), respectively. While a Bis-Tris gel was used to see the full-length APP (E), Tris-Tricine gels were used to separate APP-CTFs (F).