Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: ( a ) AKT1 and AKT2 isoforms showed decreased phosphorylation after MK2206 incubation, while AZD5363 resulted in hyperphosphorylation (AKT1: MK2206 F (3, 8) =24.80, p=0.0002, AZD F (3, 8) =37.03, p<0.0001; AKT2: MK2206 F (3, 8) =10.19, p<0.0042, AZD F (3, 8) =34.67, p<0.0001), 3 mice/group. No feedback on PDK1 was observed after incubation with AKT inhibitors (MK2206 F (3, 8) =1.665, p>0.05; AZD5363 F (3, 8) =0.636, p>0.05). ( b ) Validation of specificity of AKT1 and AKT2 phospho-antibodies. ( c ) AKT3 immunoprecipitation specifically pulls down AKT3, leaving AKT1 and AKT2 isoforms in the supernatant. Treatment with 30 µM MK2006 or AZD5363 leads to AKT3 dephosphorylation or hyperphosphorylation.

Article Snippet: Other , Brain slice incubation chamber , Scientific Systems Design Inc , BSC1 , .

Techniques: Phospho-proteomics, Incubation, Biomarker Discovery, Immunoprecipitation, De-Phosphorylation Assay

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet: Baseline recordings of field excitatory postsynaptic potentials (fEPSPs) in CA1 remain stable after incubation with MK2206 or AZD5363 (p>0.05), n = 9–11 slices/group, 4 mice/group respectively, ( F (2,6) =120.3, p<0.0001), 3 mice/group.

Article Snippet: Other , Brain slice incubation chamber , Scientific Systems Design Inc , BSC1 , .

Techniques: Incubation

Journal: eLife

Article Title: AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

doi: 10.7554/eLife.30640

Figure Lengend Snippet:

Article Snippet: Other , Brain slice incubation chamber , Scientific Systems Design Inc , BSC1 , .

Techniques: Western Blot, Immunostaining, Immunoprecipitation, Protease Inhibitor, Software, Imaging, Slice Preparation, Incubation

Journal: Nature protocols

Article Title: Near-infrared catecholamine nanosensors for high spatiotemporal dopamine imaging

doi: 10.1038/s41596-021-00530-4

Figure Lengend Snippet: a, Schematic representation of the dissection process for the generation of acute coronal brain slices. Red dashed lines represent areas to be cut by using scissors or a vibratome. b Incubation set up to label acute brain slices with nIRCat. c, Microscope perfusion bath set up to image nIRCat-labeled acute brain slices. Coronal brain slices should be placed in the perfusion bath (circle 1) and weighed down by a harp. Acute slices should be placed with the nIRCat-labeled side facing toward the microscope objective.

Article Snippet: Slice incubation chamber (Scientific Systems Design, Inc., BSK4).

Techniques: Dissection, Incubation, Microscopy, Labeling

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 1. SARS-CoV-2 3CLpro is released from infected cells by cell lysis and secreted by an unconventional protein-secretion mechanism (A) Immunoblots of 3CLpro precipitated by 33% trichloroacetic acid (TCA) from the conditioned media or in lysates from mock-infected or A549-ACE2 cells 48 hpi with SARS-CoV-2 (MOI 4, n = 5, N = 2). stds, 3CLpro standards. Densitometry of 3CLpro relative to 10 ng 3CLpro standard. (B) 3CLpro, b-tubulin, and GAPDH immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media or cell lysate from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi with MOI 0.1 or 1.0. Dotted line, intracellular 3CLpro/tubulin ratio in MOI 1.0 cell lysate (n = 3/group). (C) Immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media compared to matched lysates from SARS-CoV-2-infected Vero76 cells 48 hpi (MOI 1) or mock-infected cells (n = 3/group). 3CLpro ppt, 10 ng of 3CLpro precipitated from cell-naive media as a control; std, 10 ng of 3CLpro standard. (D) Immunoblots of 3CLpro in conditioned media concentrated by ultrafiltration and cell lysates from HEK293 cells transfected with 3CLpro expression plasmid for 48 h (n = 8, N = 2). (E) LDH activity in conditioned medium collected from 3CLpro transfected HEK293 cells (48 h) compared with the maximum amount of LDH released from lysed HEK293 cells (n = 6, N = 2). Absorbance was measured at 490 nm with 680 nm correction. (F) Immunoblots and densitometry of 3CLpro in concentrated conditioned medium and cell lysates from HEK293 cells transiently transfected with plasmids encoding 3CLpro or catalytic inactive 3CLpro Cys145Ala for 48 h (n = 5, N = 2). (G) Immunoblots and densitometry of 3CLpro or IFN-l1 after 24 h culture ± Golgi inhibitor 2 mM monensin (n = 6, N = 2). (H) 3CLpro, GSDMD, and b-actin loading control immunoblots and GSDMD densitometry of lysates from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi, MOI 0.1 or 1.0 (n = 3/group). (I) Schematic of candidate mechanisms of 3CLpro release from SARS-CoV-2-infected cells. Antibodies: anti-3CLpro (1:1,000, in-house), anti-b-tubulin (1:2,000, clone: BT7R), anti-b-actin (1:2,000, ab8226), anti-GAPDH (1:2,000, clone: GA1R), anti- GSDMD N-terminal antibody (1:500, clone: W18029B), and anti-FLAG M2 (IFN-l1 in G) (1:1,000, F3165). Data are shown as mean ± SD; statistical analysis of two groups was performed by two-tailed unpaired t test, three or more groups by one-way ANOVA with Tukey’s post hoc test, with a significance threshold of p < 0.05.

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Infection, Lysis, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 2. SARS-CoV-2 3CLpro cleaves GSDMD at LQ29Y30SS to block GSDMD pore formation, whereas caspase cleavage generates func- tional GSDMD pores that directly secrete 3CLpro and nucleocapsid protein (A) Schematic of 3CLpro cleavage sites (non-prime side [P] in blue, prime side [P0] in red) and the caspase cleavage site (violet) in human GSDMD. Y, scissile bond.

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Blocking Assay

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 3. SARS-CoV-2 3CLpro is secreted through caspase-activated GSDMD and GSDME pores and retains activity in serum to dampen platelet activation (A) Recombinant 3CLpro (2 mM) was incubated with or without recombinant GSDME (3 mg) for 2 h at 37C and resolved on 4%–12% reducing SDS-PAGE stained with Coomassie (N = 2). 3CLpro with C-terminal 33FLAG-Myc-63His tag was used. (legend continued on next page)

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Activity Assay, Activation Assay, Recombinant, Incubation, SDS Page, Staining