Journal: The Journal of Neuroscience
Article Title: Conformational Dynamics of Transmembrane Domain 3 of Presenilin 1 Is Associated with the Trimming Activity of γ-Secretase
doi: 10.1523/JNEUROSCI.0838-19.2019
Figure Lengend Snippet: Alteration of the hydrophilic environment around TMD3 is associated with Aβ42 production. A, Conditioned media of DKONL cells transiently transfected with PS1 carrying a point mutation or cotransfected with FLAG-Pen-2 were analyzed by sandwich ELISA. Aβ42 ratios [percentage of Aβ42/(Aβ40 + Aβ42)] were normalized by that of cells expressing PS1wt (A1,A2: n = 4; mean ± SEM; ***p < 0.001, compared with control by Tukey's test; A3: n = 4; mean ± SEM; *p < 0.05, compared with control by Student's t test; A4: n = 6; mean ± SEM; **p < 0.01, compared with control by Student's t test). B, AICD and NICD production by PS1 mutant or PS1/FLAG-Pen-2 was measured by luciferase assay. Measured fluorescence was normalized by the expressing level of each PS1; then data were normalized by that of wt (n = 4; mean ± SEM). *p < 0.05, compared with control (Tukey's test). **p < 0.01, compared with control (Tukey's test). ***p < 0.001, compared with control (Tukey's test). C, SCAM analysis of L166C or F177C single-Cys mt PS1 with a point mutation by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead and asterisk represent PS1 NTF and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). *p < 0.05, compared with control (Tukey's test). ***p < 0.001, compared with control (Tukey's test). D, SCAM analysis of L166C or F177C single-Cys mt PS1 with a point mutation in TMD3 by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead represents PS1 NTF. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). **p < 0.01, compared with control by Tukey's test for L166C mt and Student's t test for F177C mt. ***p < 0.001, compared with control by Tukey's test for L166C mt and Student's t test for F177C mt. E, SCAM analysis of L166C or F177C single-Cys mt PS1 FLAG-Pen-2 by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5 and anti-FLAG-tag antibody anti-FLAG M2. Gray arrowhead and asterisk represent the target proteins and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown at the right side (n = 4; mean ± SEM; not significant, compared with control by paired Student's t test). F, SCAM analyses of L166C and F177C single-Cys mt PS1 by MTSEA-biotin were performed after preincubation with ST1120. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead and asterisk represent PS1 NTF and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). *p < 0.05, compared with DMSO (paired Student's t test). NS, not significant.
Article Snippet: The anti-FALG tag antibody, monoclonal anti-FLAG M2, was purchased from Sigma-Aldrich.
Techniques: Transfection, Mutagenesis, Sandwich ELISA, Expressing, Luciferase, Fluorescence, Western Blot, Labeling, FLAG-tag
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Cell Signaling Technology Inc manufactures this product 
![Alteration of the hydrophilic environment around TMD3 is associated with Aβ42 production. A, Conditioned media of DKONL cells transiently transfected with PS1 carrying a point mutation or cotransfected with <t>FLAG-Pen-2</t> were analyzed by sandwich ELISA. Aβ42 ratios [percentage of Aβ42/(Aβ40 + Aβ42)] were normalized by that of cells expressing PS1wt (A1,A2: n = 4; mean ± SEM; ***p < 0.001, compared with control by Tukey's test; A3: n = 4; mean ± SEM; *p < 0.05, compared with control by Student's t test; A4: n = 6; mean ± SEM; **p < 0.01, compared with control by Student's t test). B, AICD and NICD production by PS1 mutant or PS1/FLAG-Pen-2 was measured by luciferase assay. Measured fluorescence was normalized by the expressing level of each PS1; then data were normalized by that of wt (n = 4; mean ± SEM). *p < 0.05, compared with control (Tukey's test). **p < 0.01, compared with control (Tukey's test). ***p < 0.001, compared with control (Tukey's test). C, SCAM analysis of L166C or F177C single-Cys mt PS1 with a point mutation by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead and asterisk represent PS1 NTF and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). *p < 0.05, compared with control (Tukey's test). ***p < 0.001, compared with control (Tukey's test). D, SCAM analysis of L166C or F177C single-Cys mt PS1 with a point mutation in TMD3 by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead represents PS1 NTF. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). **p < 0.01, compared with control by Tukey's test for L166C mt and Student's t test for F177C mt. ***p < 0.001, compared with control by Tukey's test for L166C mt and Student's t test for F177C mt. E, SCAM analysis of L166C or F177C single-Cys mt PS1 FLAG-Pen-2 by MTSEA-biotin. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5 and <t>anti-FLAG-tag</t> antibody anti-FLAG <t>M2.</t> Gray arrowhead and asterisk represent the target proteins and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown at the right side (n = 4; mean ± SEM; not significant, compared with control by paired Student's t test). F, SCAM analyses of L166C and F177C single-Cys mt PS1 by MTSEA-biotin were performed after preincubation with ST1120. Immunoblotting assays were performed using the anti-PS1 NTF antibody G1Nr5. Gray arrowhead and asterisk represent PS1 NTF and a nonspecific protein, respectively. Quantifications of the relative labeling efficiency of each PS1 are shown below (n = 3; mean ± SEM). *p < 0.05, compared with DMSO (paired Student's t test). NS, not significant.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7281/pmc06807281/pmc06807281__zns9991920250007.jpg)