Fig. 1
From: CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease
CERTL binds directly to APP and Aβ and reduces Aβ aggregation and toxicity in vitro. a Protein interaction detected using co-IP of APP and CERTL in HEK-APP. The total cell lysate of HEK-APP cells (L) lane 1 and the total cell lysate of HEK-APP cells IP using APP/Aβ (lane 2), CERTL (lane 3), and syntaxin (isotype control) (lane 4) antibodies were analyzed by Western blot. APP (1) and CERTs proteins were detected. The isotype control syntaxin protein was negative. Molecular weight markers are indicated (kDa). b Immunofluorescent staining showing co-localization of CERTs and APP/Aβ in primary neurons isolated from 5xFAD brains. Neuronal marker MAP 2 was used to immuno-label neuronal cells. DAPI was used for nuclei staining. Scale bar 5 μm. c CERTL and Aβ1–42 interaction was measured by microscale thermophoresis. The dissociation constant (Kd) calculated was 2.5 ± 0.3 μM. d Percentage of Thioflavin T (ThT) fluorescence intensity to detect Aβ1–42 20 μM aggregation in the absence or presence of recombinant CERTL 1 or 2.5 μM at different time points. Each data point represents the percentage of mean fluorescent intensity of three wells (repeated measures ANOVA; Dunnett’s multiple comparisons test **p < 0.01, ***p < 0.001). e, f TEM analysis of 2 μM Aβ1–42 aggregation in the absence and presence of 0.1 μM CERTL showed a different aggregation pattern quantified by Aβ width (Student’s t test ***p < 0.001). g Measurement of cell metabolic activity of SH-SY5Y by MTT assay in cells incubated with medium alone (control) or medium containing 10 μM Aβ1–42, Aβ1–42, and 1 μM CERTL, or CERTL alone for 24 h. Graph bar expressed as means ± S.E.M % of control N = 5–10 (one-way ANOVA, Bonferroni correction *p < 0.05; **p < 0.01). h SM d18:1/16:0, SM d18:1/18:1, SM d18:1/18:0, SM d18:1/24:1, and SM d18:1/24:0 measured by HPLC-MS/MS in N2a cells after 48-h transfection with vector control or pcDNA-CERTL. Graph bar expressed as means ± S.E.M % of control N = 4/group (one-way ANOVA, Holm-Sidak’s multiple comparisons test ***p < 0.001)