Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: Machine learning-based screening of candidate genes for DKD. ( A-B ) LASSO regression analysis identified 5 candidate genes. ( C-D ) Random forest analysis selected 10 top-ranked genes based on relative importance. ( E ) Venn diagram of the overlapping hub genes (CASP1, PRKX, TAP1) derived from LASSO regression and random forest analysis. (F) Protein-protein interaction network of hub genes generated by GeneMANIA

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques: Derivative Assay, Generated

Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: Validation of hub gene (CASP1, PRKX, TAP1) expression patterns in DKD. ( A ) Scatter plots comparing expression levels between DKD patients and controls in the GSE30122 dataset. ( B ) Heatmap of normalized expression for the three hub genes across samples. ( C-E ) Independent validation in renal tubule tissues using Nephroseq v5 database: ( C ) CASP1, ( D ) PRKX, and ( E ) TAP1. ( F-H ) Correlation of hub gene expression with glomerular filtration rate: ( F ) CASP1, ( G ) PRKX, and ( H ) TAP1. Statistical analysis: t-tests for normally distributed data (Fig. 6A, C) and Wilcoxon rank-sum tests for non-normal data (Fig. 6D, E). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, no significance

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques: Biomarker Discovery, Expressing, Gene Expression, Filtration

Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: GSEA of hub genes using GO and KEGG gene sets from MSigDB datasets. ( A-B ) GSEA results for CASP1: ( A ) GO and ( B ) KEGG analyses. ( C-D ) GSEA results for PRKX: ( C ) GO and ( D ) KEGG analyses. ( E-F ) GSEA results for TAP1: ( E ) GO and ( F ) KEGG analyses

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques:

Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: Immune infiltration analysis in DKD. ( A ) Box plot showing the relative abundance of 22 immune cell types across different groups. ( B-D ) correlation analysis between the expression of hub genes and infiltrating immune cell proportions: ( B ) PRKX, ( C ) TAP1, and ( D ) CASP1. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significance

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques: Expressing

Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: Single-cell RNA sequencing profile of kidney tissues. ( A ) UMAP projection after Harmony batch correction, integrating datasets across samples. ( B ) 22 distinct clusters visualized by UMAP plotting. ( C ) Annotation of 10 major cell types based on marker genes. ( D ) Dot plot illustrating the expression of marker genes in different cell types. ( E-G ) Bubble plots comparing expression patterns of three hub genes in PT cells (DKD vs. controls). ( E ) CASP1, ( F ) TAP1, and ( G ) PRKX. Statistical significance was determined by Wilcoxon rank-sum tests (**** P < 0.0001). LOH, loop of Henle cells; PT, proximal tubule cells; DCT, distal convoluted tubule cells; ENDO, endothelial cells; FIB, fibroblasts; LEUK, leukocytes; ICA, type A intercalated cells; PODO, podocytes; PEC, parietal epithelial cells; ICB, type B intercalated cells

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques: Single Cell, RNA Sequencing, Marker, Expressing

Journal: Inflammation

Article Title: Integrated Transcriptomic Analysis Identifies TAP1 as a Key Regulator of PANoptosis in Diabetic Kidney Disease Tubular Injury

doi: 10.1007/s10753-025-02400-7

Figure Lengend Snippet: Validation of hub genes in experimental and clinical DKD. ( A ) qRT-PCR analysis of TAP1, CASP1, and PRKX expression in HK-2 cells under low glucose (LG, 5.5 mmol/L glucose) or high glucose (HG, 25 mmol/L glucose) conditions. ( B ) Western blot analysis of TAP1 protein levels in LG or HG. ( C ) Representative kidney histology (H&E, PAS, and Masson trichrome staining) in db/m and db/db mice. Scale bar, 20 μm. ( D ) Western blot of TAP1 protein levels in kidney tissues from db/m and db/db mice ( n = 3 for each group). ( E ) Representative IHC staining of TAP1 in db/m ( n = 3) and db/db ( n = 3) mouse kidney tissue. Scale bar, 50 μm. ( F ) Representative IHC staining of TAP1 in human kidney tissues from DKD ( n = 3) and control ( n = 3) patients. Scale bar, 50 μm. * P < 0.05, ** P < 0.01; ns, no significance

Article Snippet: Paraffin-embedded kidney sections were subjected to antigen retrieval, followed by overnight incubation at 4 °C with anti-TAP1 primary antibody (1:200, Proteintech).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Western Blot, Staining, Immunohistochemistry, Control

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by GFAP + ependymo-radial glial cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of GFAP and P2X 7 labelling in naïve fish (A-C′), at 7 dpi (D-F′), and at 14 dpi (G-I′). Colocalization (arrows) was seen in radial processes and cell bodies (C′,F′,I′). GFAP + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by PCNA + proliferating cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of PCNA + and P2X 7 labelling in naïve fish (A-C′), at 7 dpi rostral (D-F′), and at 7 dpi caudal (G-I′). Colocalization (arrows) was seen in some cells (C′,F′,I′). PCNA + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by HuC/D + neurons. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of HuC/D + and P2X 7 labelling in naïve fish (A-C′), at 14 dpi rostral (D-F′), and at 14 dpi caudal (G-I′). Colocalization (arrows) was seen in larger mature neurons and smaller immature neurons (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: Temporal changes in P2X 7 protein expression following SCI in adult zebrafish. Schematic representations of naïve and injured spinal cord areas of analysis (A-B), as well as tissue collection timeline (C). Representative western blot and corresponding total protein in naïve zebrafish (Zf) spinal cord tissue, mouse (Ms) hippocampal tissue as a positive control (+), and pre-absorption of naïve zebrafish spinal cord tissue with a blocking peptide as a negative control (-) (D). Representative western blot and corresponding total protein at 1 dpi (E), 7 dpi (G), and 14 dpi (I). Quantitative analysis of the 50 kDa isoform showed significant downregulation within the injury (In, n =10, one-way ANOVA with Dunnett's post hoc test, P =0.0079) and caudal to the lesion (Ca, n =9, P =0.0046) at 7 dpi when normalized to naïve tissue ( n =9) (H). Protein expression of the 50 kDa isoform showed basal levels of expression at 1 dpi (F) and 14 dpi (J). Data presented as means±s.d. **, significant differences, P <0.01. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques: Expressing, Western Blot, Positive Control, Blocking Assay, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Schema of TRPC3/TRPC6 chimera. A, domains and motifs in the TRPC3 C terminus. B, representation of the proximal (C1) and distal (C2) C termini of TRPC3 and TRPC6. C, schematic models of TRPC3 chimeras: TRPC3-C6C1, TRPC3-C6C2, and TRPC3-C6TRP. D, schematic models of TRPC6 chimeras: TRPC6-C3C1, TRPC6-C3C2, TRPC6-C3TRP, and TRPC6-C3TRP-C3C2. For B–D, the origin of the TRP domain is shown by the color of the oval. The amino acid (AA) number at the beginning and at the end of the C1 and C2 domains contributed by TRPC3 (top) or TRPC6 (bottom) is indicated. FLAG-tagged chimeras expressed FLAG at the N terminus, and V5-tagged chimeras expressed V5 at the C terminus of the channel.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: TRP domains, leucine zipper motifs, and AMPK binding site in TRPC3 and TRPC6. A, amino acid compositions of TRP domains and leucine zipper motifs of TRPC3 and TRPC6 are shown. Letters in boldface type indicate exchanged amino acids in the chimeras. For TRP domain exchange, the boldface sequences in the TRP domain of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6TRP. The boldface amino acids of TRPC3 TRP were exchanged with those of TRPC6 to create TRPC6-C3TRP. For leucine zipper exchange, the boldface sequences in the leucine zipper of TRPC6 were exchanged with those of TRPC3 to create TRPC3-C6LZ. The boldface amino acids of TRPC3 were exchanged with those of TRPC6 leucine zipper to create TRPC6-C3LZ. B, exchange of TRPC3 741–748 and TRPC6 802–809 is shown. Amino acids contributed by TRPC3 (top) or TRPC6 (bottom) are indicated, and localization to the C1 or C2 part of the C terminus is shown.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of TRP domains in regulation of TRPC3 and TRPC6 by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC3-C6TRP, or BFP-TRPC6-C3TRP chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 17, Epo 21), BFP-TRPC6 (PBS 17, Epo 22), BFP-TRPC3-C6TRP (PBS 16, Epo 22), or BFP-TRPC6-C3TRP (PBS 18, Epo 21). The Epo-stimulated increase in cells expressing TRPC6 and Epo-R is not statistically different from cells expressing Epo-R alone and is thought to be secondary to Epo-R activation of low levels of endogenous channels (17). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Role of the distal C terminus of TRPC3 and TRPC6 in regulation by Epo-R. HEK 293T cells were transfected with BFP-TRPC3, BFP-TRPC6, BFP-TRPC6-C3TRP, BFP-TRPC6-C3C2, BFP-TRPC6-C3TRP-C3C2, BFP-TRPC3-C6 802–809, or BFP-TRPC6-C3TRP-C3 741–748 chimeras and Epo-R. Fura Red-loaded cells were treated with 40 units/ml Epo. To quantitate [Ca2+]i, F440/F490 was measured at base line and by monitoring over 20 min after Epo stimulation. Shown is the percentage increase in F440/F490 above base line (mean ± S.E. (error bars) percentage increase) = peak F440/F490 divided by base line F440/F490 × 100% − 100% (base line). The numbers of individual cells studied were as follows: BFP-TRPC3 (PBS 56, Epo 102), BFP-TRPC6 (PBS 57, Epo 103), BFP-TRPC6-C3TRP (PBS 20, Epo 34), BFP-TRPC6-C3C2 (PBS 20, Epo 35), BFP-TRPC6-C3TRP-C3C2 (PBS 30, Epo 64), BFP-TRPC3-C6 802–809 (PBS 26, Epo 49), or BFP-TRPC6-C3TRP-C3 741–748 (PBS 12, Epo 30). *, significantly greater percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC6 (p < 0.001). **, significantly less percentage increase in F440/F490 compared with Epo-stimulated cells expressing wild type TRPC3 (p < 0.001).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Plasma membrane insertion of TRPC3/TRPC6 chimeras detected with cell surface biotinylation. HEK 293T cells transfected (Tx'd) with Epo-R and V5-TRPC3, V5-TRPC3-C6TRP, V5-TRPC6, V5-TRPC6-C3TRP, V5-TRPC6-C3C2, V5-TRPC6-C3TRP-C3C2, or V5-TRPC6-C3TRP-C3 741–748 were stimulated with 0–40 units/ml Epo for 0–5 min. Biotinylation of cell surface proteins was performed, and V5-tagged proteins were immunoprecipitated (IP) from lysates with anti-V5 antibody. Western blots (WB) of immunoprecipitates were probed with streptavidin-HRP to detect biotinylated protein and then stripped and reprobed with anti-V5-HRP to detect total protein. Representative results of Western blots from three experiments are shown. Biotinylated and total protein bands were quantitated with densitometry, and the ratio was normalized to time 0. The mean ± S.E. (error bars) values of the biotinylated/total protein ratios from three experiments after 5 min of stimulation are shown. *, significant difference in the ratio compared with time 0 (p < 0.05).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Subcellular localization of TRPC3, TRPC6, TRPC3/TRPC6 chimeras, PLCγ, and Epo-R. A, proteins from HEK 293T cells transfected with FLAG-TRPC3, FLAG-TRPC3-C6TRP, FLAG-TRPC6, FLAG-TRPC6-C3TRP-C3C2, and Epo-R were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Transfected Epo-R was detected with anti-Epo-R antibody, and endogenous PLCγ was detected with anti-PLCγ antibody. Results from four fractionation experiments using FLAG-tagged constructs and two experiments using V5-tagged constructs were similar, and representative results with FLAG constructs are shown. B, representative results showing quality of fractionation by probing Western blots with anti-GAPDH, anti-Na+K+-ATPase, anti-lamin, and anti-vimentin (markers for cytosol, membrane, nuclear, and cytoskeletal fractions, respectively).

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation

Journal: The Journal of Biological Chemistry

Article Title: The Transient Receptor Potential (TRP) Channel TRPC3 TRP Domain and AMP-activated Protein Kinase Binding Site Are Required for TRPC3 Activation by Erythropoietin *

doi: 10.1074/jbc.M111.238360

Figure Lengend Snippet: Membrane and cytoskeletal association of TRPC3, TRPC6, and TRPC3/TRPC6 chimeras. Proteins from transfected HEK 293T cells (A–C) or UT-7/Epo cells (C) were fractionated, purified, and analyzed by Western blotting (WB). Transfected FLAG-tagged constructs were detected by probing with anti-FLAG antibody. Quality of fractionation was confirmed by probing Western blots with anti-Na+K+-ATPase (membrane), anti-vimentin (cytoskeletal fraction), and anti-GAPDH (cytosol marker) antibodies. Equivalent input from lysates was confirmed by probing with anti-actin. A, fractionation with the Qiagen cell fractionation kit. Membrane and cytoskeletal association of TRPC3/6 channels and chimeras. Three to nine experiments (depending on the FLAG-tagged construct) were performed, and representative results are shown. B, fractionation using the 0.5% Triton X-100 extraction method. Representative results of three experiments are shown. C, subcellular fractionation of endogenous TRPC3 and TRPC6 in UT-7/Epo cells with the Qiagen fractionation kit. For all preparations, 100 μg/lane was loaded except for the cytoskeletal fraction of transfected cells, where 50 μg was loaded per lane. Endogenous TRPC3 was detected using anti-TRPC3-C antibody. Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody. Transfected HEK 293T cells were used as controls. Representative results of four experiments are shown. lys, whole cell lysate; M, membrane fraction; Csk, cytoskeletal fraction. *, bands that did not disappear with peptide blocking, indicating that they are nonspecific.

Article Snippet: Endogenous TRPC6 was detected using Alomone anti-TRPC6 antibody.

Techniques: Transfection, Purification, Western Blot, Construct, Fractionation, Marker, Cell Fractionation, Blocking Assay

Journal:

Article Title: Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes

doi: 10.1111/j.1469-7793.2001.0339c.xd

Figure Lengend Snippet: A, amplified DNA fragments of P2X receptors (lanes 1–7) from rat brain (a) and rat portal vein myocytes (b) were separated on a 2 % agarose gel and visualized by staining with ethidium bromide. Lane 8, RNA from brain and portal vein myocytes in the absence of reverse transcriptase served as a negative control. Numbers on the left indicate molecular size standards in base pairs (bp). For RNA purification and PCR conditions, see Methods. B, immunostaining of P2X receptor subtypes in portal vein myocytes. Myocytes were stained with anti-P2X1 receptor (a) or anti-P2X4 receptor antibody (b) and vizualization was realized with a donkey anti-rabbit IgG FITC-conjugated antibody. In the absence of primary antibodies or after inactivation of the antibodies by their antigen peptides, only a faint background fluorescence was observed (not shown). Typical confocal sections were performed above the nucleus and therefore appeared spherical. Both P2X1 (a) and P2X4 (b) receptor subtypes were distributed throughout the confocal sections with a marked staining of P2X1 receptor subtype at the cell periphery.

Article Snippet: The rabbit anti-P2X1 and anti-P2X4 receptor antibodies (Alomone Labs, Jerusalem, Israel) were directed against polypeptides corresponding to residues 382–399 and 370–388 of the rat P2X1 and P2X4 receptors, respectively.

Techniques: Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Purification, Immunostaining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex

doi: 10.3390/ijms221910457

Figure Lengend Snippet: Human cortical spheroids express the serotonin transporter SERT. ( A ) 3-month-old untreated cortical spheroids were immunostained with the SERT antibody or ( B ) the SERT antibody together with the blocking peptide to evaluate antibody specificity. Endogenous β-actin-GFP (green) and immunostained SERT (magenta) were imaged with confocal microscopy under identical conditions. Scale bar = 10, 20 μm.

Article Snippet: Antibodies toward serotonergic proteins were also used: SERT (Alomone Lab, Jerusalem, Israel, AMT-004), SERT blocking peptide (Alomone Lab BLP-MT004), 5-HT2A (Sigma Aldrich SAB4501474) and 5-HT5A (Sigma Aldrich SAB4501483.

Techniques: Blocking Assay, Confocal Microscopy

Journal: The Journal of Neuroscience

Article Title: Presynaptic L-Type Ca 2+ Channels Increase Glutamate Release Probability and Excitatory Strength in the Hippocampus during Chronic Neuroinflammation

doi: 10.1523/JNEUROSCI.2981-19.2020

Figure Lengend Snippet: Long-lasting LPS treatment increases the expression of L-type Ca2+ channels at glutamatergic synapses. A, Top, Representative images of neurites of hippocampal neurons exposed to either vehicle or LPS for 48 h and double-immunostained with Cav1.2 antibodies (green) and the excitatory presynaptic marker vGLUT1 (red). Merge panels represent synaptic puncta in which Cav1.2 and vGLUT1 colocalize (yellow; arrows). Bottom, Representative immunoreactivity intensity profiles of the colocalization experiments showing the increased occurrence of overlap between Cav1.2- and vGLUT1-positive puncta in the samples chronically treated with LPS. B, Quantification of the density of vGLUT1-positive puncta counted on 30 μm dendrite tracts starting from the neuronal body. Data are mean ± SEM from three independent experiments, each conducted in duplicate (veh = 30; LPS = 29). C, Quantification of the absolute Cav1.2/vGLUT1 colocalization area (top) and of the Cav1.2/vGLUT1 colocalization expressed in percent of the total Cav1.2-immunopositive area (bottom) in vehicle- and LPS-treated neurons (veh = 66; LPS = 97). Data are mean ± SEM. **p < 0.01 (unpaired Student's t test/Mann–Whitney U test).

Article Snippet: The presynaptic localization of the L-type Ca 2+ channel was analyzed in treated neurons by evaluating the immunoreactivity of L-type Ca 2+ channel (1:200, Alomone Labs) ( Hermosilla et al., 2017 ) in vGLUT1-positive excitatory boutons.

Techniques: Expressing, Marker, MANN-WHITNEY