anti-tgn38 nbp1-03495  (Novus Biologicals)

Journal: Frontiers in Physiology

Article Title: Tmem45b modulates itch via endoplasmic reticulum calcium regulation

doi: 10.3389/fphys.2025.1708686

Figure Lengend Snippet: Tmem45b is essential for the calcium regulation of ER. (A) Representative immunostaining images show the colocalization between Tmem45b (green) and TGN38 (red) in the DRG tissue sections. Scale bar, 20 μm. (B) The volcano plot illustrates the significant differences between WT and CKO mice (WT vs. cKO, n = 3 vs. 4), assessed based on the fold change (cKO/WT) and adjusted p-value (padj). Blue dots represent genes that are significantly downregulated (fold change <0.67; padj <0.1), red dots indicate significantly upregulated genes (fold change >1.5; padj <0.1). Genes represented by black dots show no significant differences between the groups. (C) qPCR analysis. (WT vs. cKO, n = 4 vs. 4). Columns represent the mean expression level of Atp2a1 mRNA normalized to Gapdh . (D) Western blot analysis. Experiments are repeated three times. (E) Schematic of calcium imaging in dissociated DRG neurons response to thapsigargin. (F) Representative calcium imaging response to thapsigargin (100 μM). (G) Statistical results. (WT vs. cKO, n = 90 vs. 72). (H) Schematic of calcium imaging in dissociated DRG neurons in response to caffeine (10 mM). (I) The heatmap shows the calcium activity of DRG neurons in response to two rounds of caffeine stimulations. Neuron numbers are shown on the left of the image. (J) Statistical results. (K) Schematic of calcium imaging in dissociated DRG neurons response to Trypsin (500 nM). (L, M) Representative calcium response to Trypsin. (N) Statistical results. (WT vs. cKO, n = 227 vs. 217). Two-tailed unpaired Student’s t test. DRG was obtained from at least three mice. Data are expressed as the Mean ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies used in this study: GM130 (BD, 610822), Th (Millipore, AB1542), IB4 (Vector, FL-1201-.5), Tuj1 (Starter, SDT-251-28), CGRP (Dia Sorin, 24112), NF200 (CST, 2836S), PDI (Santa Cruz, SC-20132), Calnexin (Abcam, ab112995), Serca1 (Proteintech, 22361-1-AP), mitochondria-tracer (Beyotime, C1048), TGN38 (Bio-Rad, AHP499G), GFAP (Millipore, MAB3402), IBA1 (Abcam, ab5076).

Techniques: Immunostaining, Expressing, Western Blot, Imaging, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Clustering of NLRP3 induced by membrane or protein scaffolds promotes inflammasome assembly

doi: 10.1038/s41467-025-60277-4

Figure Lengend Snippet: a Conserved basic segments within the linker and FISNA region of NLRP3 homologs. b Immunoblot of non-localized and organelle-tethered NLRP3-YFP with respective NLRP3 mutants K127A/K128A/K129A/K130A (4xA). c – h Confocal images of NLRP3-KO iBMDMs stably transduced with organelle-tethered NLRP3-YFP (yellow) variants 12 h post priming with or without nigericin stimulation (Nig), stained for nuclei (Hoechst 33342, cyan) and plasma membrane (Cholera Toxin Subunit B, CTB, magenta). Scale bar, 10 µm. Following priming, 10 µM nigericin, 0.2 mg/ml SiO 2 or 20 ug/ml imiquimod (Imq) were added to cells, respectively, as indicated in the respective graph columns with legends above. Measurements of IL-1β were normalized to each respective non-mutated, organelle-enriched NLRP3-YFP: WT ( c ), Cb5 ( d ), TGN38 ( e ), HRas ( f ), Rheb ( g ) or Omp25 ( h ) treated with canonical stimuli: nigericin, SiO 2 or imiquimod, respectively. EV, empty vector. Data represents the mean ± SEM of three independent experiments, except for TGN38, treated with SiO 2 in ( e ), which is the mean ± SEM of two independent experiments. The average value calculated from technical replicates within an individual experiment is presented as a single data point. P values were calculated using two-tailed unpaired t -test with Welch’s correction ( c – h ). Western blots ( b ) and microscopic images ( c – h ) are representative of three independent experiments. Panel ( a ) created in Biorender. Hafner Bratkovic, I. (2025) https://BioRender.com/99kdc99 .

Article Snippet: Localization sequences were amplified from plasmids encoding Hras (a gift from D. Sabatini, Addgene 26637 ), Tmem192 (a gift from D. Sabatini, Addgene 102930 ), Rheb (a gift from D. Sabatini, Addgene 26634 ), TGN38 (a gift from J. Lippincott-Schwartz, Addgene 128148), GOLGB1 (a gift from D. Gadella, Addgene 67903 ), Omp25 (a gift from D. Sabatini, Addgene 26638 ), PEX3 (a gift from J. Boehm & W. Hahn & D. Root, Addgene 81779 ), PEX26 (a gift from D. Sabatini, Addgene 139054 ) and PACT (a gift from C. Norden, Addgene 105954 ).

Techniques: Western Blot, Stable Transfection, Transduction, Staining, Clinical Proteomics, Membrane, Plasmid Preparation, Two Tailed Test