Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in western blot analysis

Article Snippet: PSN2 rabbit polyclonal , 1:800 , 16168-1-AP , Proteintech.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Modulation of AD proteins by miR-502-3p. (A) Immunoblot analysis of amyloid precursor protein (APP), tubulin associated unit (Tau), presenilin-1 (PSN1) and presenilin-2 (PSN2) and -actin protein in hippocampal neurons (HT22) cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The levels of AD proteins were changed in miR-502-3p agomiR and antagomiR transfected cells. (B) Densitometry analysis of APP, Tau, PSN1 and PSN2 blot shows increased protein levels in miR-502-3p agomiR transfected cells compared with scramble control, while the cells treated with miR-502-3p antagomiR shows a reduced level of all AD proteins compared with agomiRs. For statistical analysis, Student’s t -test was used for analyzing the data between three groups of samples such as scramble control, agomiRs, and antagomiRs. Experiments were performed in triplicates. The P -values < 0.05 were considered statistically significant. AD: Alzheimer’s disease; miR: microRNA.

Article Snippet: PSN2 rabbit polyclonal , 1:800 , 16168-1-AP , Proteintech.

Techniques: Western Blot, Transfection, Control

Journal: Molecular Neurobiology

Article Title: Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-Amyloid Assemblies

doi: 10.1007/s12035-021-02567-8

Figure Lengend Snippet: Contribution of presenilins to the production of hexameric Aβ assemblies. a SH-SY5Y knockdown (KD) cell lines were generated using CRISPR-Cas9, with guide RNA vectors targeting either human PSEN1 (PS1-KD) or PSEN2 (PS2-KD) genes. Control cells were transfected with respective scrambled sequences. Left, a representative Western blot; middle and right, quantitative decrease in PS1 and PS2 protein levels in KD compared to S cells (for all conditions, see Supplementary Fig. ). N = 3. One-sample t test with hypothetical value set as 100: * p < 0.05, *** p < 0.001 (S vs KD, in PS1: p = 0.03; in PS2: p = 0.0001). WT wild-type, S scrambled. b , c Initial cleavage ability was monitored by a reporter gene assay. The release of APP intracellular domain (AICD) from a tagged C99-GVP substrate was measured by the Gal4- Firefly reporter gene. Results are represented as Firefly / Renilla luciferases ratios, with Renilla serving as a transfection-efficiency control. The profile of Aβ production was assessed after transfection with either an empty plasmid (EP) or C99, using Western blotting and ECLIA immunoassay, in PS1-KD vs PS1-S (in b ) and in PS2-KD vs PS2-S (in c ). Media samples were lyophilized prior to analysis. Dashed lines indicate that proteins were run on the same gel, but lanes are not contiguous. Luciferase assays (initial cleavage of C99): N = 4 each. One-way ANOVA with Tukey's multiple comparison test: ns = non-significant (S vs KD, in PS1 control: p > 0.99; in PS1 C99-GVP: p = 0.10; in PS2 control: p = 0.99; in PS2 C99-GVP: p = 0.99). Western blots quantitative analyses (hexameric Aβ, in cell lysates and released, relative to C99 and expressed as a % to S): N = 3 each. One-sample t test with hypothetical value set as 1: ns non-significant, ** p < 0.01 (S vs KD, in PS1 cell lysates: p = 0.16; in PS1 media: p = 0.97; in PS2 cell lysates: p = 0.99; in PS2 media: p = 0.01). ECLIA assays (monomeric Aβ released): N = 5 each. Mann–Whitney test: ns non-significant, * p < 0.05 (S vs KD, in PS1 Aβ 40 : p > 0.99; in PS1 Aβ 42 : p > 0.99; in PS2 Aβ 40 : p = 0.04; in PS2 Aβ 42 : p = 0.12)

Article Snippet: Kits each containing guide RNA vectors that target human PSEN1 or PSEN2 genes, a GFP–puromycin or RFP–blasticidin donor vector, respectively, and a scrambled sequence control were obtained from Origene (CAT#: KN216443 and KN202921RB).

Techniques: Knockdown, Generated, CRISPR, Control, Transfection, Western Blot, Reporter Gene Assay, Plasmid Preparation, Luciferase, Comparison, MANN-WHITNEY

Journal: Molecular Neurobiology

Article Title: Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric β-Amyloid Assemblies

doi: 10.1007/s12035-021-02567-8

Figure Lengend Snippet: PS2-dependent release of hexameric Aβ assemblies in extracellular vesicles (EVs). Complete media of PS1-S, PS1-KD, PS2-S, and PS2-KD cells underwent an ultracentrifugation process to separate putative enrichment of EVs, in pellet, from soluble proteins. a The efficiency of EVs isolation was confirmed by plate-based Europium-immunoassay (left panel) showing an enrichment of EVs inclusion markers CD9, CD63, and CD81, while EVs exclusion marker GM130 was lower in EVs as compared to soluble fractions. N = 6. Mann–Whitney test: * p < 0.05, *** p < 0.001 (CD9 ( n = 22), CD63 ( n = 22), and CD81 ( n = 18): p < 0.0001; GM130 ( n = 19): p = 0.0362). Quantification of protein levels by BCA (right panel) showed larger protein amounts in soluble than EVs fractions, ruling out the specific enrichment of inclusion markers due to higher content in proteins. b EVs ultracentrifugation pellets were counted for number of particles of 70-400 nm by nanoparticle tracking analysis (NTA). n = 9 in N = 3 independent experiments. Two-way ANOVA with Bonferroni’s multiple comparison: * p < 0.05 (PS2-KD EP vs PS2-S EP: p < 0.05). c Both EVs and soluble extracts were monitored by Western blotting with the W0-2 antibody. Dashed lines indicate that proteins were run on the same gel, but lanes are not contiguous. d Human PS2 was re-expressed in PS2-KD cells to assess the restoration of observed phenotypes. The efficiency of re-expression was monitored using the PS2-CTF antibody in PS2 rescued (R) cells with comparison to PS2-S and PS2-KD cells (left panel). Cell lysates (middle panel) as well as extracellular soluble proteins (upper right panel) and EVs (lower right panel) were monitored by Western blotting with the anti-Aβ (W0-2) antibody. EP empty plasmid, EV(s) extracellular vesicle(s), Sol soluble proteins fraction

Article Snippet: Kits each containing guide RNA vectors that target human PSEN1 or PSEN2 genes, a GFP–puromycin or RFP–blasticidin donor vector, respectively, and a scrambled sequence control were obtained from Origene (CAT#: KN216443 and KN202921RB).

Techniques: Isolation, Marker, MANN-WHITNEY, Comparison, Western Blot, Expressing, Plasmid Preparation