cbh v5 aav9 abe c terminus  (Addgene inc)

Journal: bioRxiv

Article Title: Identification of Human Transferrin Receptor as an Entry Co-receptor for Parvovirus B19 Infection of Human Erythroid Progenitor Cells

doi: 10.64898/2026.04.02.715920

Figure Lengend Snippet: (A) Protein diagram. VP1u-APEX2 consists of APEX2 fused to the C-terminus of the unique region of B19V VP1 (VP1u) via a seven-residue glycine-serine linker (GGSGGSG), followed by a Flag tag and a 6 × Histidine (His) tag. APEX2 has a linker-Flag-His tag fused at the C-terminus. (B) Analysis of purified proteins. VP1u-APEX2 and APEX2 proteins were expressed in bacteria and purified. Approximately (∼) 1 µg of each protein was separated by SDS-PAGE, followed by Coomassie blue staining. M, molecular weight marker. (C) Confocal microscopy of VP1u-APEX2 entry. 1 × 10 6 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C for 2 h. The cells were then immunostained with α-Flag to visualize internalized proteins under a Leica STED confocal microscope. Scale bar = 10 μm. Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole). (D) Western blotting of APEX2-biotinylated proteins. 1 × 10 7 UT7/Epo-S1 cells were incubated with 2 μM VP1u-APEX2 or APEX2 protein at 37°C. After 2 h, APEX2-mediated biotinylation was then performed as described in the Materials and Methods and Figure S1 . Biotinylated host proteins were purified with streptavidin-conjugated magnetic beads. The supernatant was collected as the flow-through (FT), and the beads were further washed several times and eluted as the pull-down (PD). Both FT and PD samples were analyzed by SDS-PAGE and immunoblotting using Alexa Fluor 680-conjugated streptavidin. (E) Analysis of VP1u-APEX2-biotinylated/associated proteins using quantitative mass spectrometry (qMS). Three independent PD samples prepared from VP1u-APEX2 and APEX2 (control) treated cells were analyzed by on-bead digestion and qMS. MS data were processed and analyzed as described in the Materials and Methods. The bubble plot shows protein enrichment (log 2 fold change) in the VP1u-APEX2 group relative to the APEX control, with color indicating subcellular localization based on Gene Ontology (GO) annotation. TFRC denotes human transferrin receptor 1 (hTfR).

Article Snippet: Purified proteins: Recombinant hTfR ECD protein tagged with a His-tag at the C-terminus (#11020-H07H) and recombinant human ferritin heavy chain 1/FTH1 (#13217-HNAE) were purchased from SinoBiological (Paoli, PA).

Techniques: Residue, FLAG-tag, Purification, Bacteria, SDS Page, Staining, Molecular Weight, Marker, Confocal Microscopy, Incubation, Microscopy, Western Blot, Magnetic Beads, Mass Spectrometry, Control, Protein Enrichment

Journal: Journal of Advanced Research

Article Title: HDAC2 enhances the antimicrobial activity of neutrophils by promoting the formation of neutrophil extracellular traps (NETs) in sepsis

doi: 10.1016/j.jare.2025.08.041

Figure Lengend Snippet: HDAC2 enhances antimicrobial activities against E.coli in mice. (A) Bacterial loads in the blood were calculated in HDAC2-knockout and wild type mice. (A)The picture of the bacterial clones was taken by BIO-RAD ChemiDoc“MP Imaging System. (B)The number of colony-forming units (CFU) in the blood of mice was calculated. HDAC2 WT and HDAC2 KO mice were administrated with E. coli- GFP (5 × 10 7 ) by intravenous Injection. Blood (20 μl) was taken from the tail vein of the mice, applied to the pretreated solid MHA medium, and incubated at 37 °C for 20 h. Count the colonies on the culture medium and take pictures. The experiments were performed in quintuplicate, data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). (B) Bacterial loads in the kidneys were calculated in HDAC2-knockout and wild type mice. The picture of kidneys from HDAC2 WT and HDAC2 KO mice were taken. The number of colony-forming units (CFU) in the kidneys of mice was calculated. The kidneys from HDAC2 KO and HDAC2 WT mice were weighed, ground, spread on the MHA plates, incubated for 16–24 h at 37 °C, and then bacterial clones were counted. The experiments were performed in quintuplicate, data are presented as the means ± SEM. of independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t -test). n = 5 mice per group. (C) H&E staining of livers and kidneys. The arrows indicate damage section of tissue. (D) Serum CRP and PCT levels of mice with E. coli- GFP bacteremia at 24 h. The CRP and PCT were measured by ELISA. Data are shown as mean ± SEM. *p < 0.05; n = 5 mice per group. ns, not significant.

Article Snippet: Concentrations of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), histone deacetylase 2 (HDAC2), PCT, CRP and myeloperoxidase (MPO) in mouse serum were quantified using their respective enzyme-linked immunosorbent assay (ELISA) kits, following the protocols provided by the manufacturer (Boster Biological Technology, Wuhan, China).

Techniques: Knock-Out, Clone Assay, Imaging, Injection, Incubation, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy

doi: 10.1016/j.xcrm.2026.102639

Figure Lengend Snippet: Intrathoracic ABE treatment corrects Tnni3 R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual AAV9 vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3 R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3 R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗ p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following the AAV9-ABE treatment. As in (C), gene editing efficiency in the livers of Tnni3 R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

Article Snippet: The plasmids Cbh_v5 AAV9-ABE N-terminus (Addgene plasmid no. 137177) and Cbh_v5 AAV9-ABE C-terminus (Addgene plasmid no. 137178) were employed for AAV plasmid construction.

Techniques: Mutagenesis, Plasmid Preparation, Next-Generation Sequencing, Expressing