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: Journal of Neurochemistry

Article Title: Validation of TRPA1 and TRPV1 Antibodies for Expression Detection in Mammalian Cells and Tissues

doi: 10.1111/jnc.70444

Figure Lengend Snippet: Calcium imaging and immunocytochemistry showing TRPV1 expression in transfected HEK‐293 cells. (A) Fura2 calcium imaging in HEK‐293 cells transiently expressing rTRPV1‐EYFP fusion protein. Representative traces of calcium transients evoked by capsaicin in EYFP + cells. Green traces correspond to EYFP + cells and gray traces to EYFP − cells. Pie chart represents the percentage of EYFP + cells that responded or failded to respond to capsaicin. Data obtained from 88 cells from 3 coverslips. (B–F left) Confocal images of TRPV1‐EYFP transiently expressed in HEK‐293 cells. EYFP (green), TRPV1 antibody (magenta) and Hoechst (HO, blue). Merge images correspond to the overlap between TRPV1 and EYFP on the left and Hoechst and brightfield (BF) on the right. Antibody dilution of the representative images is indicated in the lower right corner. Scale bar: 20 μm. (B–F right) Box plots display the specificity ratio (SR) for each tested dilution of the corresponding antibody. Each dot represents a single EYFP + cell. Boxes indicate the interquartile range (25th to 75th percentiles) and whiskers extend to the 5th and 95th percentiles. The horizontal line inside each box marks the median and the black dot indicates the mean. (*** p < 0.001 Mann–Whitney test). (G) Bar histogram graph summarizes the SR median difference between the tested antibody dilution and their controls lacking primary antibody (secondary antibody only; rabbit, goat or guinea pig) as calculated with the Hodges‐Lehman estimator. Error bars represent the 95% confidence interval. For each antibody and dilution, a minimum of 100 cells were analyzed across 4 fields from 2 independent transfections. $ Santa Cruz V1 antibody was generated against a human TRPA1 peptide.

Article Snippet: Only the Abcam and Neuromics GP antibodies successfully detected TRPV1 (Figure , full uncropped blots in Figure ), consistent with their superior efficiency in ICC of TRPV1‐transfected cells (Figure ).

Techniques: Imaging, Immunocytochemistry, Expressing, Transfection, MANN-WHITNEY, Generated

Journal: Journal of Neurochemistry

Article Title: Validation of TRPA1 and TRPV1 Antibodies for Expression Detection in Mammalian Cells and Tissues

doi: 10.1111/jnc.70444

Figure Lengend Snippet: Western blot analysis for TRPV1 and TRPA1 antibodies specificity. (A) Immunoblots for TRPV1 using Abcam (1:1000), Millipore V1 (1:1000), Santa Cruz V1 (1:100), Neuromics GT (1:200), and Neuromics GP (1:1000) antibodies. (−) lanes: Untransfected HEK‐293 cells, (+) lanes: HEK‐293 cells transfected with rTRPV1‐EYFP (B) Immunoblots for TRPA1 using Aviva (1:300), Novus (1:500), Millipore A1 (1:500), Alomone (1:200), Proteintech (1:500), Sigma WH (1:500), and Santa Cruz A1 (1:100). (−) lanes: Untransfected HEK‐293 cells, (+) lanes: HEK‐293 cells transfected with hTRPA1‐tGFFP. For each antibody, top row: Immunoblot with each TRPV1/TRPA1 antibody. Middle row: EYFP/tGFP immunoblotting of the same membrane. Bottom image: GAPDH loading control. Each blot was performed at least three times to rule out technical artifacts in cases where no anti‐TRPV1/TRPA1 signal was detected. For every repetition, the identical lysate sample was probed with all antibodies. Full uncropped blots are available in the Figure . $ Santa Cruz V1 antibody was generated against a human TRPA1 peptide.

Article Snippet: Only the Abcam and Neuromics GP antibodies successfully detected TRPV1 (Figure , full uncropped blots in Figure ), consistent with their superior efficiency in ICC of TRPV1‐transfected cells (Figure ).

Techniques: Western Blot, Transfection, Membrane, Control, Generated

Journal: Journal of Neurochemistry

Article Title: Validation of TRPA1 and TRPV1 Antibodies for Expression Detection in Mammalian Cells and Tissues

doi: 10.1111/jnc.70444

Figure Lengend Snippet: Immunocytochemistry of endogenously expressed TRPV1 in DRG cultures from rTRPV1‐EGFP transgenic mice. (A–D left): Confocal images of TRPV1 + DRG neurons incubated with the indicated antibody. EGFP (green), TRPV1 antibody (magenta) and βIII‐Tubulin (cyan). Scale bar: 50 μm. (A–D right) box plots display the specificity ratio (SR) for each tested dilution of the corresponding antibody. Each dot represents a single EGFP + cell. Boxes indicate the interquartile range (25th to 75th percentiles) and whiskers extend to the 5th and 95th percentiles. The horizontal line inside each box marks the median and the black dot indicates the mean. (*** p < 0.001 Mann–Whitney test). (E) Bar histogram graph summarizes the SR median difference between the tested antibody dilution and their controls lacking primary antibody (secondary antibody only; rabbit, goat or guinea pig) as calculated with the Hodges‐Lehman estimator. Error bars represent the 95% confidence interval. For each antibody and dilution, a minimum of 92 cells were analyzed across 4 microscopic fields from 2 different animals.

Article Snippet: Only the Abcam and Neuromics GP antibodies successfully detected TRPV1 (Figure , full uncropped blots in Figure ), consistent with their superior efficiency in ICC of TRPV1‐transfected cells (Figure ).

Techniques: Immunocytochemistry, Transgenic Assay, Incubation, MANN-WHITNEY

Journal: Journal of Neurochemistry

Article Title: Validation of TRPA1 and TRPV1 Antibodies for Expression Detection in Mammalian Cells and Tissues

doi: 10.1111/jnc.70444

Figure Lengend Snippet: Immunohistochemistry of endogenously expressed TRPV1 in DRG slices from rTRPV1‐ EGFP transgenic mice. (A–D left): Confocal images of TRPV1 + DRG neurons incubated with the indicated antibody. EGFP (green), TRPV1 antibody (magenta) and βIII‐Tubulin (cyan). Scale bar: 50 μm. (A–D right) box plots display the specificity ratio (SR) for each tested dilution of the corresponding antibody. Each dot represents a single EGFP + cell. Boxes indicate the interquartile range (25th to 75th percentiles) and whiskers extend to the 5th and 95th percentiles. The horizontal line inside each box marks the median and the black dot indicates the mean. (* p < 0.05, *** p < 0.001 Mann–Whitney test). (E) Bar histogram graph summarizes the SR median difference between the tested antibody dilution and their controls lacking primary antibody (secondary antibody only; rabbit, goat or guinea pig) as calculated with the Hodges‐Lehman estimator. Error bars represent the 95% confidence interval. For each antibody and dilution, a minimum of 141 cells were analyzed across 4 pictures from 2 different animals.

Article Snippet: Only the Abcam and Neuromics GP antibodies successfully detected TRPV1 (Figure , full uncropped blots in Figure ), consistent with their superior efficiency in ICC of TRPV1‐transfected cells (Figure ).

Techniques: Immunohistochemistry, Transgenic Assay, Incubation, MANN-WHITNEY

Journal: Journal of Neurochemistry

Article Title: Validation of TRPA1 and TRPV1 Antibodies for Expression Detection in Mammalian Cells and Tissues

doi: 10.1111/jnc.70444

Figure Lengend Snippet: Comparison of immunofluorescence of endogenously expressed TRPV1 in cultured cells and slices from TRPV1‐KO (KO) and TRPV1‐EGFP mice. (A–C) Immunocytochemistry. (D–F) Immunohistochemistry. (A–) Confocal images of cultured DRG neurons and DRG tissue sections (D–F) from TRPV1‐KO (KO, left) and TRPV1‐EGFP (right). EGFP (green), TRPV1 antibody (magenta), and βIII‐Tubulin (cyan). Scale bar: 50 μm. (A–F) For each antibody and dilution, 4 pictures from 2 different animals were studied, image display settings are the same for KO and TRPV1‐EGFP.

Article Snippet: Only the Abcam and Neuromics GP antibodies successfully detected TRPV1 (Figure , full uncropped blots in Figure ), consistent with their superior efficiency in ICC of TRPV1‐transfected cells (Figure ).

Techniques: Comparison, Immunofluorescence, Cell Culture, Immunocytochemistry, Immunohistochemistry