Fig. 2: ELK1 promoted APP amyloidogenic processing by inhibiting the ubiquitination and degradation of PS1. | Experimental & Molecular Medicine

Fig. 2: ELK1 promoted APP amyloidogenic processing by inhibiting the ubiquitination and degradation of PS1.

From: ELK1 inhibition alleviates amyloid pathology and memory decline by promoting the SYVN1-mediated ubiquitination and degradation of PS1 in Alzheimer’s disease

Fig. 2

a ELK1 or ELK1 small hairpin RNA (shELK1) plasmid was transfected to N2AAPP cells. a–j ELK1 (a, b), APP (a, c), BACE1 (a, d), PS1 (a, e–g), NCT (a, h), APH1A (a, i) and PEN2 (a, j) were determined by western blot (WB) 48 h later (n = 3–7 in each group). *P < 0.05 and ***P < 0.001, determined by one-way ANOVA. k ELK1 or shELK1 plasmid was transfected to 2EB2 cells. PS1 was determined by WB 48 h later (n = 5 in each group). *P < 0.05 and **P < 0.01, determined by one-way ANOVA. l The N2AAPP cells were exposed to MG132 (10 μM) or CQ (50 μM). PS1 was determined by WB 24 h later (n = 7 in each group). ***P < 0.001, determined by one-way ANOVA. m The HEK293 cells were treated with or without MG132. PS1-Flag together with or without Ub-HA plasmid was transfected to these cells 6 h later. PS1 ubiquitination was evaluated 24 h later by immunoprecipitation (IP) with antibodies to Flag or negative control antibodies (IgG) and immunoblotting (IB) with antibodies to HA (n = 3 in each group). n PS1-Flag together with Ub-HA or HA-tagged ubiquitin mutants (K6R, K11R, K27R, K29R, K33R, K48R and K63R) was transfected to HEK293 cells. PS1 ubiquitination was evaluated by coimmunoprecipitation (Co-IP) as described above (n = 2 in each group). o–q PS1-Flag together with Ub-HA or HA-tagged ubiquitin mutants K27 (o), K48 (p) and K63 (q) was transfected to HEK293 cells. PS1 ubiquitination was evaluated by Co-IP as described above (n = 2–3 in each group). r PS1-Flag or Flag-tagged PS1 mutants (K311, K314, K380, K395, K429 and K430) was transfected to HEK293 cells. The ubiquitination of PS1 and PS1 mutants were evaluated 24 h later by IP with antibodies to Flag or IgG and IB with antibodies to ubiquitin (n = 4 in each group). s The N2AAPP cells were transfected with or without ELK1, followed by exposure to CHX (100 μg ml−1) for the indicated time. PS1 was determined by WB to evaluate its degradation (n = 8 in each group). ***P < 0.001, determined by two-way ANOVA. t PS1-Flag and Ub-HA, along with or without ELK1, were transfected to HEK293 cells. PS1 ubiquitination was evaluated by Co-IP as described above (n = 3 in each group). ns, not significant.

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