paper n a recombinant dna plasmid pqe60 qiagen  (Qiagen)

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: Cells

Article Title: C-Terminus of Ca v 1.3 L-Type Ca 2+ Channel Upregulates Its Own Gene Expression

doi: 10.3390/cells15090828

Figure Lengend Snippet: The cleaved form of the Cav1.3-C-terminus is localized within the nucleus. ( A ) Schematic representation of the Cav1.3 α1 subunit with the four domains (I–IV), six repeat segments (1–6), pore region (P), N-terminus (brown), intracellular loops (IC, green) and the C-terminus (C-term, blue) with the relevant regulatory domains (Isoleucine-glutamine (IQ); EF-Helix binding motif (EF hand)). The commercial anti-II-III loop and anti-C-terminus antibodies’ epitope are shown in red. Anti-Ca v 1.3-C-terminus antibody labeling shows green fluorescent staining of the nucleus and the cytoplasm in ( B ) non-infected neonatal rat myocytes (NRVMs), ( E ) non-infected adult rat atrial myocytes (ARAMs) and ( H ) Ca v 1.3-transfected tsA201 cells. DAPI in blue ( C , F ) and red ( I ) is a nuclear staining marker. Arrows indicate nuclear staining. Overlay images are shown in ( D , G , J ). The scale bar is 20 μm. ( K ) Surface plot illustrating the staining intensity of the C-terminus in the nucleus and cytoplasm using the anti-C-terminus antibody. The central gray peak corresponds to nuclear green fluorescence. ( L ) Representative Western blots of nuclear and cyptoplasmic fractions from non-infected NRVMs and ARAMs probed with the C-terminus antibody (n = 5). Histone Deacetylase (HDAC) and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) were used as internal controls.

Article Snippet: Briefly, cardiac cells were fixed, permeabilized, blocked, and incubated overnight at 4 °C with a primary anti-C-terminus antibody (Pacific Immunology, Ramona, CA, USA) targeting a unique epitope (amino acids 1708–1724) in the C-terminus of Ca v 1.3 and detected with FITC-conjugated anti-rabbit antibody (1:200).

Techniques: Binding Assay, Antibody Labeling, Staining, Infection, Transfection, Marker, Fluorescence, Western Blot, Histone Deacetylase Assay

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