Journal: Cell death and differentiation

Article Title: Notch signaling in response to excitotoxicity induces neurodegeneration via erroneous cell cycle reentry.

doi: 10.1038/cdd.2015.23

Figure Lengend Snippet: Figure 1 Cell cycle reentry following KA insult is associated with nuclear Notch signaling. (a) Representative fluorescence immunohistochemistry photomicrographs of primary neuronal cultures transfected with a Ccnd1:CDK4 (4D) construct colabeled with act-Casp3 and DAPI. Arrow indicates a binucleate 4D+ neuron. (b) Pie chart summarizing the proportion of neurons labeled with GFP only, those colabeled with act-Casp3 or those colabeled with act-Casp3 and with binucleate morphology (n = 50 cells). (c) Representative immunofluorescence photomicrograph of Ccnd1 expressing neuron from the WT hippocampi treated with KA. (d) Quantitation of the percentage of Ccnd1- positive neurons in Saline and KA hippocampi (n = 4 WT mice per condition). (e) Hippocampal CA1 layer, 2 days following KA insult, shows the presence of BrdU+ neurons expressing Notch1 in the nucleus (white arrow and insert). (f) Graph summarizing the proportion of BrdU+ cells as a percentage of NeuN+ neurons in the CA layer following KA as compared with Saline controls (n = 951 cells from n = 4 mice per condition). (g) Representative immunoblot shows the extent of cleaved Notch1 in hippocampal lysates at 12 h after KA as compared with Saline controls. (h) Optical density quantitation of the NICD1 bands 12 h after Saline and KA injection (n = 8 per condition). (i) Representative immunoblot on immunoprecipitated hippocampal lysates using an antibody against the cytoplasmic tail of Notch1 shows the following interactions- Notch1:Kpna6 and Notch1: fbw7, and ubiquitination of Notch1 in Saline and KA conditions. No trace of protein is visible in the control IP sample with serum. GAPDH is used to control for loading of the inputs and contamination in the IP samples (n = 4 independent IPs). (j) Graph summarizing the expression of the Notch targets; Hes1, Hey1 and C-myc, upon KA in WTand RBPJKcKO hippocampi as compared with the respective Saline controls (n = 4-8 mice per condition). Asterisks indicate significant differences. Error bars represent mean ± S.E.M. Scale bars in A and C is 50 μm and E is 100 μm

Article Snippet: Following antibodies were used for immunoblot in this study: rabbit anti Notch1 (1 : 1000; 07-220, Upstate/ Millipore, Darmstadt, Germany), mouse anti Notch1 (mN1A; 1 : 250; SAB4700742, Sigma-Aldrich), rabbit anti cleaved N1, NICD1 (1 : 1000; #2421, Cell Signaling), rabbit anti Importin7 (Kpna6; 1 : 1000; ab105350, Abcam), mouse anti Fbw7 (1 : 1000; NBP1-59631, Novus, Cambridge, UK), mouse anti PSD95 (1 : 500; sc-32290, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti Ubiquitin (1 : 1000; sc-8017, Santa Cruz Biotechnology), rabbit anti PTEN (1 : 500; ab154812, Abcam), mouse anti Akt (1 : 1000; sc-5298, Santa Cruz Biotechnology), rabbit anti phospho-Akt (1 : 1000; #4060, Cell Signaling), mouse anti GSK3β (1 : 1000; NBP1-47470, Novus), rabbit anti phospho-GSK3β (1 : 1000; ab107166, Abcam), rabbit anti-RBPJK (1 : 1000; #5313, Cell Signaling), mouse anti Rb (1 : 100; #MS595P, Thermo Fisher Scientific), rabbit anti phospho-Rb (1 : 1000; orb14852, Biorbyt, Cambridge, UK), mouse anti Ccnd1 (1 : 200; MA5-12702, Thermo Fisher Scientific), rabbit anti E2F1 (1 : 500; ab112580, Abcam), mouse anti GAPDH (1 : 5000; 1D4, Novus) and mouse anti β-Actin (1 : 2000; sc-8432, Santa Cruz Biotechnology).

Techniques: Fluorescence, Immunohistochemistry, Transfection, Construct, Labeling, Immunofluorescence, Expressing, Quantitation Assay, Saline, Western Blot, Injection, Immunoprecipitation, Ubiquitin Proteomics, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Ehrlichia chaffeensis Tandem Repeat Effector Targets Differentially Influence Infection

doi: 10.3389/fcimb.2017.00178

Figure Lengend Snippet: E. chaffeensis TRP-interacting host proteins involved in cell apoptosis .

Article Snippet: Other antibodies used in this study were mouse anti-human FBXW7 (R&D Systems, Minneapolis, MN), α-tubulin and PTPN2 (Santa Cruz, Dallas, TX), and rabbit anti-human CD14 (Abgent, San Diego, CA), EIF3A, ERA1, KARS, LMAN1, and STAT6 (Proteintech, Rosemont, IL), FYN and SLC43A3 (Santa Cruz), IRF2BP2 and SEPX1 (Pierce, Rockford, IL), KDM6B (Novus, Littleton, CO), and RC3H1 (Sigma).

Techniques: Binding Assay, Modification, Histone Deacetylase Assay

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: Representative sequencing electropherogram of the ANXA4 variant (p.G8D). The arrow indicates the location of the mutation, and patient “RSA-219” harbored the ANXA4 p.G8D variant (A) . Evolutionary conservation analysis of ANXA4 p.G8D in vertebrate species (B) .

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: Structural differences between wild-type ANXA4 (p.G8) and mutated ANXA4 (p.D8) proteins. The protein structures of the wild-type and mutated ANXA4 proteins were modeled based on the crystal model of the human ANXA4 protein.

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques:

Journal: Frontiers in Medicine

Article Title: Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion

doi: 10.3389/fmed.2024.1462649

Figure Lengend Snippet: The ANXA4 p.G8D mutation inhibited cell migration, invasion and adhesion in stably transfected THESCs. THESCs were cultured and transfected with ANXA4 wild-type (WT) or mutant-type (MT) plasmids, while pcDNA3.1 was used as a control plasmid (Con). Twenty-four hours after transfection, G418 was used to screen stably transfected cells, and one individual clone was separated by dilution. ANXA4 expression in stably transfected cells was confirmed by WB (A,B) and RT–qPCR (C) . The effects of ANXA4 mutation on cell migration (D,F) and invasion (E,G) were determined by Transwell assays, and cell adhesion (H,I) was determined by Matrigel adhesion assays. These assays revealed that ANXA4 p.G8D mutation inhibited cell migration, invasion and adhesion in stably transfected cells compared with ANXA4 WT. Scale bars, 100 μm. The data are shown as the means ± SDs. p < 0.05 was considered to indicate a significant difference.

Article Snippet: The membranes were subsequently incubated with the following primary antibodies at 4°C overnight: anti- ANXA4 (1:1000, TA803051; Origene, China) and anti- β -actin (1,2000, sc-47778; Santa Cruz).

Techniques: Mutagenesis, Migration, Stable Transfection, Transfection, Cell Culture, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: STAR Protocols

Article Title: Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9

doi: 10.1016/j.xpro.2022.101978

Figure Lengend Snippet:

Article Snippet: FBXW7/Cdc4 Antibody (1:100) , Novus Biologicals , Cat# H00055294-M02.

Techniques: Recombinant, Software, Cell Culture, Transferring, Membrane, Microscopy, Transfection