Journal: Annals of clinical and translational neurology

Article Title: Clinical heterogeneity within the ALS-FTD spectrum in a family with a homozygous optineurin mutation.

doi: 10.1002/acn3.52075

Figure Lengend Snippet: Figure 4. (A–C): Rt-PCR revealed OPTN-mRNA expression in PBMCs from the brother of the index patient (listed as Pat2, A). On a protein level, optineurin could not be detected in PMBC lysates (B) or lysates from postmortem frontal cortex of the index patient (listed as Pat1) in western blot analysis (C). (D–I) Anti-phospho-TDP-43 immunohistochemistry on spinal cord sections of a sporadic case of ALS (D–F) and of the index patient ALS and the optineurin mutation (G–I). Phospho-TDP43-positive neuronal skein-like and compact cytoplasmic inclusions in motor neurons in the control case (D) are also immunopositive (arrows) for two different anti-optineurin antibodies (E: non-C-terminal; F: C-terminal). In contrast, in the patient with the optineurin mutation, the inclusions (G) do not immunoreact with any of the two anti-optineurin antibodies (H and I). Scale bars: 50 lm: D–F; 100 lm: G–I;

Article Snippet: The Trans-Blot TurboTM Transfer System was used for transfer of proteins onto nitrocellulose membranes at 25 V for 15 min. Membranes with loaded proteins were incubated with EveryBlot Blocking Buffer (Bio-Rad) at room temperature for 30–60 min, followed by incubation with primary antibodies at 4°C for at least 8 h. We used two OPTN polyclonal antibodies (Proteintech, Cat. No. 10837-1-AP, at a dilution of 1:10,000 & Cayman, Item No. 100000, at a dilution of 1:1000), of which one binds to the C-terminus of optineurin (Cayman), and a GAPDH antibody (Cell Signaling, Cat No. 2118, at a dilution of 1:1000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry, Mutagenesis, Control

Journal: Theranostics

Article Title: Alisol A ameliorates vascular cognitive impairment via AMPK/NAMPT/SIRT1-mediated regulation of cholesterol and autophagy

doi: 10.7150/thno.112661

Figure Lengend Snippet: Modulation of NAMPT activity and its network by Alisol A restores cholesterol balance, mitophagy, and synaptic function in Ldlr -/- mice. (A) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B protein quantitation for WB, n = 3. (B) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B in cells, and quantification result was normalized against the levels of GAPDH or COXIV, n = 3; (C) Changes in MMP was monitored by staining with JC1 in HT22 cells, scale: 100 μm; The ratio of red/green fluorescence intenity was employed to assess MMP, n = 8; (D) Representative IF images of OPTN and TOM20 in HT22 cells, scale: 2 μm; Quantification of IF stain intensity using Image J, n = 8; IF and colocalization analysis. (E) WB analysis of PINK1 in HT22 cells and quantification result was normalized against the levels of GAPDH, n = 3; (F) Subsequent COIP experiments employed NAMPT antibodies to assess interactions with NAMPT and PINK1, using IgG as a control. (G) Illustrative IF images depicting hippocampal neurons located within the CA3 subregion, scale: 100 μm, enlarge scale: 10 μm, n = 5; IF was quantified for UCP2 (red); (H) The phenomenon of intercellular ROS generation was observed through the detection of intracellular ROS levels using the DCFH-DA probe following different treatment protocols, scale: 100 μm, n = 5; (I) Intracellular ROS levels were measured using flow cytometry, comparative analysis of fluorescence intensity within peak plots derived from flow cytometry, n = 5; (J) Synapses in the hippocampal CA1 and DG region were revealed by IF staining for the pre-and postsynaptic markers, Synapsin-1 (red) and PSD95 (green) respectively, scale: 10 μm, enlarge scale: 2 μm, n = 5; and quantification of IF; (K) Golgi-Cox Staining Hippocampal Golgi-Cox staining was performed as previously described, scale: 100 μm, enlarge scale: 20 μm, n = 8; Track the neural branches after Golgi staining and analysis of Sholl results and statistical data; Analysis of dendritic spine density in neurons stained with Golgi, along with the quantification of mushroom spines, scale: 2 μm; (L) Representative IF images of NMDA 2A (green) and NMDA 2B (red), scale: 2 μm; and quantification of IF, n = 5. Compared to the Ldlr -/- mice , * P < 0.05, ** P < 0.01; compared to the FK866 group, # P < 0.05, ## P < 0.01. Compared to the 1, * P < 0.05, ** P < 0.01; compared to the 3, # P < 0.05, ## P < 0.01. DMSO: HT22 +DMSO; Rosup: HT22 + Aβ 25-35 (20 μM) and complex (50 μg/ml of cholesterol) +H 2 O 2 0.1 mM ; CCCP: HT22+ CCCP 10 μM. Compared to the 5, * P < 0.05, ** P < 0.01; compared to the 7, # P < 0.05, ## P < 0.01.

Article Snippet: Anti-GFAP antibody (GB12096, Servicebio, 1:500), anti-IBA-1 antibody (GB12105, Servicebio, 1:500). anti-PSD95 antibody (20665-1-AP, Proteintech, 1:300), anti-SYNAPSIN-1 antibody (20258-1-AP, Proteintech, 1:300), anti-UCP2 antibody (GB11377, Servicebio, 1:300), anti-CD68 antibody ( GB113109 , Servicebio, 1:300), anti-α-SMA antibody ( GB111364 , Servicebio, 1:300), anti-HMGCR antibody (DF6518, Affinity, 1:200), anti-ABCA1 Polyclonal antibody (26564-1-AP, Proteintech, 1:300). anti-NAMPT/PBEF Polyclonal antibody (11776-1-AP, Proteintech, 1:300), anti-SIRT1 antibody (13161-1-AP, Proteintech, 1:300). anti-CYP46A1 Polyclonal antibody (124861-1-AP, Proteintech, 1:300). anti-OPTN antibody ( GB114324 , Servicebio, 1:300), anti-TOM20 antibody ( GB111481 , Servicebio, 1:300), anti-NMDAR2A/GRIN2A Polyclonal antibody (28525-1-AP, Proteintech, 1:300), anti-NMDA2B antibody (21920-1-AP, Proteintech, 1:300).

Techniques: Activity Assay, Protein Quantitation, Staining, Fluorescence, Control, Flow Cytometry, Derivative Assay