anti rat antisera  (Vector Laboratories)

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) Experimental scheme of scRNA-seq analysis of epithelial cells. Infant mice (P5) (n=3/group) were infected with V. cholerae and sacrificed 20 hours post-infection (hpi). The proximal and distal SI were collected, and epithelial cells were isolated. Single-cell RNA libraries were prepared from sorted Epcam+ cells. (b) UMAP of the distal small intestine epithelial single-cell RNA-seq data from uninfected and V. cholerae infected P5 infant mice (n=3 for each condition, cells were pooled); EEC, enteroendocrine cells. The arrow indicates cluster 5 enterocytes in infected animals in the distal SI. ( c) Top-hit pathways from the Single Cell Pathway Analysis (Qval > 8.75 in any cluster). Dot plot represents enrichment in color and Qval in circle size. Stem, stem cells, EC, enterocytes, Gob, Goblet cells, EEC, enteroendocrine cells. ( d) Volcano plot showing q-value (Qval) and pathway enrichment from Single Cell Pathway Analysis. Pathway enrichment indicates a mean pathway change comparing uninfected and V. cholerae -infected conditions. Colors represent the following pathways: IFN (red), TNFα and Inflammatory (yellow), Mitosis (green), Translation and Ribosome (light blue), Golgi, ER, and mTOR (pink), and Metabolism (dark blue). ( e) Percentage of cells in each cluster vs the total cell number in the distal SI. ( f) GO pathway analysis of cluster 5 representative genes. (g) Fgb and Sult6b2 gene detection by single molecule RNA FISH co-stained with DAPI (blue) in the distal SI tissue at 20 hpi. White arrowheads in the higher magnification image indicate the expression of Fgb (green) and Sult6b2 (magenta) mRNA in epithelial cells. Dotted line indicates the epithelial cell membrane. Signal intensity was adjusted to visualize single puncta in the high magnification image. Color-separated images are shown in Extended Data. Fig.3b. ( h) Representative immunostaining of Reg3β (red), V. cholerae (magenta), and DAPI (blue) in the distal SI from an uninfected animal (left) or V. cholerae -infected animal (right). Scale bar: 100 μm. White arrows show the colocalization of Reg3β and V. cholerae on the epithelium. ( i) Deconvoluted z-stack image of V. cholerae (green) and Reg3β (red) from a puncta of Reg3β/ V. cholerae colocalization image. Overlay image was taken by confocal microscope using x100 lens and z-stack with 0.3 micron gaps per layer. (j) Pearson’s correlation coefficient between Reg3β and V. cholerae co-localization using the images of ( h ) from infected animals. 55 images from 4 animals were analyzed and plotted in a violin plot. ( is partially created in https://BioRender.com .)

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: Infection, Isolation, RNA Sequencing, Staining, Expressing, Membrane, Immunostaining, Microscopy

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) UMAP of CD45+ immune cells (23,836 cells including all conditions: proximal and distal SI, mock and infected samples) in the lamina propria of the small intestine. (b) Heatmap of the numbers of differentially expressed genes (absolute Fold Change > 1, adjusted p-value < 0.00001, genes on chromosome X and Y were excluded as both, male and female infant mice were used for each condition, in each cluster). ( c) Dot plots showing enrichment and Qval of the Single Cell Pathway Analysis results in color and circle size, respectively. Clusters with more than 500 cells (total cell number) isolated from distal SI are shown. (d ) Volcano plots showing enrichment and Qval of Single Cell Pathway Analysis results in the representative clusters for B cells, T cells, and ILC3s. Colors represent pathways “Interferon” for IFN (red), Proinflammatory, IL17, and IL23 (green), Translation and Ribosome (blue), Golgi, ER, and mTOR (purple). (e) CellChat -based analysis of up-regulated signaling pathways from LTi-like ILC3 to enterocytes. Il22-Il22Ra1/Il10Rb signaling is highlighted. Color code represents the types of enterocytes in the receiver. (f) Il22+ cell populations in Cd45+ cells in lamina propria (LP, n = 3-4/group) and intraepithelial lymphocyte (IEL, n = 5-6/group). Data are represented as mean ± SD for biological replicates. Unpaired t-test. Data are representative of two independent experiments. (g) Whole mount staining of Il22 (yellow) and Epcam (green) in uninfected or infected distal SI. Images are representative of three independent samples/group. (h) qPCR analysis of cluster 5 enterocyte marker gene ( Fga , Fgb , and Sult6b2 ) expression in the distal SI from V. cholerae infected/uninfected WT C57BL/6 (Il22 +/+ ), infected Il22 +/− and Il22 −/− littermate animals. Expression levels were normalized to β-Actin expression and shown as relative expression to uninfected Il22 +/+ group.

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: Infection, Isolation, Protein-Protein interactions, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) Schematic of IL22 prophylactic regime. A barcoded library of V. cholerae was used in these experiments to enable measurement of the founding population (Ns), the number of cells from the inoculum that establish infection, and CFU/Ns which represents the net replication of the founders during infection. (b-d) CFU (b), founding population (Ns) (c), and replication (CFU/Ns) (d) of V. cholerae in the proximal and distal SI. Vehicle: PBS-treated animals, IL22Fc: IL22Fc-treated animals. Bars indicate the geometric mean for biological replicates (n = 5-6/group). Unpaired t-test. ( e) Kaplan-Meyer plot of infant mice survival in V. cholerae infection in IL22Fc-treated or control animals; vehicle control (black, n = 6), IL22Fc-treated group (red, n = 8). Log-rank test. ( f) Percent weight gain relative to the time of infection; vehicle (black, n = 6), IL22Fc-treated group (red, n = 8). Data are represented as mean ± SD. Two-way ANOVA. ( g) Diarrhea in V. cholerae -infected infant mice at 48 hpi. White arrowheads in vehicle-treated animals point to clear diarrheal fluid on the anus. (h) V. cholerae burden in the proximal and distal SI at the time of death (vehicle-treated, black, n = 6) or 60 hpi (IL22Fc-treated, red, n = 8). Bars indicate the geometric mean. Unpaired t-test. P-values are shown.

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: Infection, Control

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) Experimental scheme of the transposon-insertion sequencing screen. Mice were given vehicle (PBS, n = 3) or 5μg of IL22Fc (n = 4) at 24 hours before infection and were infected with 10 8 cells of a V. cholerae transposon library. SI tissues were harvested at 20 hpi. (b) XY plot of transposon insertion frequency compared to inoculum (culture on LB plates); x-axis represents the mean of log 2 fold change (bacteria recovered from distal SI of PBS-treated mice vs. inoculum) and y-axis represents the mean of log 2 fold change (bacteria recovered from distal SI of IL22Fc-treated mice vs. inoculum). Purple dots: Tcp-biogenesis mutants; green dots: Flagellar-related mutants. The plot represents the mean value of biological replicates (Control: n = 3, IL22Fc: n = 4). c) Heatmap representing log 2 fold change of transposon insertion frequency in flagellar genes in samples recovered from mouse tissue compared to inoculum. Column and row show individual mouse and V. cholerae genes, respectively. The magma color heatmap indicates the difference in the average of log 2 fold change between IL22Fc-treated mice vs. control mice (d) Competitive index (CI) using barcoded mutant vs. WT strains in vehicle (black, n = 8) and IL22Fc-treated animals (red, n = 7). Multiple unpaired t-tests were performed between control vs IL22Fc-treated animals. Bars indicate the geometric mean, with the dotted line showing CI = 1. (e ) CFU of V. cholerae detected in the luminal wash and attached to the tissue of the distal SI in vehicle-treated animals (black, n = 3) and IL22Fc-treated animals (red, n = 4). ( f ) Ratio of bacterial CFU in luminal washes compared with CFU of tissue-associated bacteria in vehicle-treated (black, n = 3) or IL22Fc-treated animals (red, n = 4). Unpaired t-test. ( g) Representative images of WT V. cholerae colonization in the crypt in control animal (left image) and IL22Fc-treated animal (right image), and a bar graph showing the percent of crypts occupied by V. cholerae in the distal SI. Blue: DAPI, red: V. cholerae LacZ::tdTomato , magenta: phalloidin for actin staining. Scale bar: 25 μm. P-values are shown in individual figures.

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: Sequencing, Infection, Bacteria, Control, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) UMAP of epithelial single-cell RNA-seq data of the distal small intestine in P5 infant mice in control and IL22Fc-treated animals. ( b) Comparison of cluster abundance between IL22Fc-treated and untreated control animals. ( c) Dot plot representing control vs. IL22Fc-treated gene expression changes of cluster 5 cell marker genes (Fig. S3A) in epithelial cell subsets. Color represents average log 2 fold change of gene expression compared to untreated control. Dot size represents negative log 10 -scaled adjusted p-value. ( d) V. cholerae growth curve measured by OD600 after incubation with native or heat-inactivated Reg3β (left). Percent V. cholerae survival after incubation with indicated concentration of Reg3β (right). Data represent mean ± SD for experimental replicates; native Reg3β (n = 4/condition), heat-inactivated Reg3β (n = 2/condition). ( e-g) CFU (e), founding population (Ns) (f), and CFU/Ns (g) of V. cholerae in the proximal and distal SI at 16 hpi. Infant mice were orally administered 2 doses of vehicle (PBS, n = 4) or Reg3β recombinant protein (33 μg/dose, n = 5) at −0.5 and +1 hpi. Bars represent the geometric mean for biological replicates. One-way ANOVA. ( h-j) CFU (g), Ns (h), and CFU/Ns (i) of V. cholerae in the proximal and distal SI of P5 Reg3β-/- mice and heterozygous littermate at 16 hpi. Bars represent the geometric mean for biological replicates. Open circles indicate no CFU detected. One-way ANOVA. Sample groups were compared among the same tissue. P-values are shown.

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: RNA Sequencing, Control, Comparison, Gene Expression, Marker, Incubation, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

doi: 10.1101/2025.09.12.675873

Figure Lengend Snippet: (a) Violin plot of traditional marker gene expression in stem cells, secretory progenitor cells, and goblet cells. Lgr5 and Olfm4 : stem cell marker, Mki67 ; proliferating cell marker, Atoh1 and Spdef ; secretory progenitor cell marker, Muc2 : Goblet cell marker, Lyz1 : Paneth cell marker. ( b) Heatmap of goblet and Paneth cell marker expression in adult (Haber et al: GSE92332( 16 )) and IL22Fc-treated P5 infant mice. ( c) Representative RNAScope images of Atoh1 (green) and Spdef (magenta) expression in the epithelial cells in the crypts of the distal small intestine of vehicle- and IL22Fc-treated infant mice. ( d, e) The number of Atoh1 + (d), or Spdef + (e) cells in the SI epithelium. Vehicle: n = 122 images from 4 animals, IL22Fc: n = 141 images from 4 animals. Unpaired t-test. ( f) Representative images of immunohistochemistry of E-Cadherin (red), Muc2 (green), and DAPI (blue) in the distal small intestine of vehicle- and IL22Fc-treated animals. White arrowheads point to the Muc2+ cells in the crypts. ( g) PAS staining of the distal small intestine of vehicle and IL22Fc treated animals. Arrowheads indicate PAS+ cells in the crypt. Vehicle: n = 63 images from 3 animals, IL22Fc: n = 65 images from 3 animals. ( h) Enumeration of PAS+ cells in the crypts. Unpaired t-test with Welch’s correction. ( i ) Western blot image of SI luminal wash detected with anti-Muc2 antibody. n = 5/group. (i) Appearance of luminal washes collected from control and IL22Fc-treated infant mice (left). Turbidity of luminal washes were measured as OD600 in a spectrophotometer (right). (j) Immunoprecipitation-western blot for Muc2 in luminal washes (right blot) and the signal intensity analyzed by densitometry (right bar graph). Signal intensity was normalized by the average intensity in control samples. n =5/group. ( k-m) CFU (k), FP (l), and replication (CFU/FP) (m) of V. cholerae in the small intestine of P5 infant Muc2 −/− vs littermate wild type or heterozygous controls (Muc2 +/+, +/− ) animals treated with (+) or without (−) IL22Fc. n = 6-28 animals/group. Bars represent the geometric mean for biological replicates. One-way ANOVA. Sample groups were compared among the same tissue. P-values are indicated in individual plots. Open circles indicate no CFU detected.

Article Snippet: Tissue sections were washed in PBS, blocked in blocking buffer [5% goat serum 0.1% Triton X100 PBS] for 30 min, and stained with rabbit anti-Fga (1:100, ProteinTech), rabbit anti-Fgb (1:100, ProteinTech), sheep anti-Reg3β antibody (1:100, R&D Systems), rabbit anti- V. cholerae antisera Ogawa (1:100, BD Difco), and Ulex Europaeus Agglutinin I (UEA-I) Fluorescein (1:100, Vector) at 4 °C overnight.

Techniques: Marker, Gene Expression, Expressing, RNAscope, Immunohistochemistry, Staining, Western Blot, Control, Spectrophotometry, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: AKAP18 recruits ubiquitin specific proteinases. A , validation and proteomic identification of AKAP18 interactors in neonatal cardiomyocytes. Immunoblot shows the expression and biotinylation patterns of GFP-miniTurbo (lanes 1 and 2) and AKAP18-miniTurbo (lanes 3 and 4). Cells were treated ± biotin. Biotinylated proteins were detected using streptavidin-HRP. Representative image is shown from three independent experiments. Molecular weight markers are indicated. B , molecular function gene ontology groups for AKAP18 (SS ≥ 0.7). C , STRING analysis (Ver 12.0) associated with PKA binding (GO:0051018) and ( D ) ubiquitin-like protein ligase binding (GO:0044389) clusters. E , plasmids encoding FLAG-tagged AKAP18γ-mini turbo was coexpressed with empty vector (pCDNA3) or plasmids encoding MYC-tagged USP4 or MYC-tagged USP7 in HEK293 cells. Expression of USP (lanes 3 and 4) and AKAP18γ (lanes 2, 3, and 4) was confirmed by immunoblot analysis of whole-cell lysates, with β-actin serving as a loading control ( left panel ). Complex formation was assessed by pull-down of USPs using anti-MYC antibody and detection by Western blot of FLAG-tagged AKAP18γ (lanes 3 and 4, middle panel ). F , quantification of AKAP18γ complex formation with USPs from three independent experiments by densitometric analyses is presented in the graph form. Data shown as the mean ± SEM from three biological replicates. Statistics: paired t test using nonnormalized values, ∗∗ p < 0.01, ∗ p < 0.05. AKAP18, A-kinase anchoring protein 18; HRP, horseradish peroxidase; PKA, protein kinase A; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Ubiquitin Proteomics, Biomarker Discovery, Western Blot, Expressing, Molecular Weight, Binding Assay, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: Validation of AKAP18 interaction with USP4. A , proximity ligation assay (PLA) shows ( left ) control, ( mid ) RII-AKAP18 and ( right ) USP4-AKAP18 puncta in representative mouse adult cardiomyocytes. B , quantification of PLA puncta per 1000 μm 2 from five cells of each condition using Fiji/ImageJ. C , IgG control (lane 1) and USP4 (lane 2) immune complexes isolated from HMC cells. Immunoblot detection of ( top ) SERCA2, ( upper-mid ) USP4 and ( lower mid ) PKAc. Immunoblot detection of USP4 ( bottom ) serves as a loading control. Molecular weight markers are indicated. D , immunofluorescence detection of USP4 ( green ), AKAP18 ( magenta ) and SERCA2 ( cyan ) in paraformaldehyde fixed section of human heart tissue. E , composite staining pattern. The scale bar represents 20 μm. (Inset) composite imaging showing codistribution of three signals ( white ) at higher magnification. All measurements presented as means ± SEM. AKAP18, A-kinase anchoring protein 18; IgG, immunoglobulin G; PKAc, protein kinase A catalytic subunit; PLA, proximity ligation assay; SERCA2, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Biomarker Discovery, Proximity Ligation Assay, Control, Isolation, Western Blot, Molecular Weight, Immunofluorescence, Staining, Imaging, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: The central domain of AKAP18 interfaces with USP4. A , schematic of AKAP7 gene organization depicting exon–intron structure and alternatively spliced isoforms. Exon 7 ( cyan ) encodes the PKA anchoring domain. B and C , AKAP18 γ and δ isoforms bind USP4. GFP tagged AKAP18 isoforms were expressed with V5 tagged USP4 in HEK293 cells. B , immunoblot detection of ( top panel ) V5-USP4 and ( lower panel ) AKAP18 isoforms in GFP immune complexes. Immunoblot detection of IgG controls (lanes 1, 3, 5, 7, and 9) are included. Molecular weight markers are indicated. C , activity measurements of immune complexes by AMC assay (RFU). IgG fractions ( gray ) and GFP ( charcoal ) are indicated. Deubiquitinase activities above background ( green ) are specified. Data from three experiments is presented. C , coomassie blue staining of ( left ) purified Flag-USP4 and ( mid ) GST and GST-AKAP18δ proteins. AKAP18δ pull-down of USP4 is visible ( mid panel , lane 8). ( Right panel ) AKAP18δ pull-down of USP4 confirmed by immunoblot detection (lane 12). E and F , chemical cross-linking studies and molecular modeling of the USP4/AKAP18 interface. E , chemical cross-linker BDP (concentrations indicated above each lane) incubated with purified USP4 with the central (CD) domain of AKAP18. Coomassie blue stained gel revealing cross linked protein complexes. USP4, AKAP18 CD and protein complex are indicated ( F ) Amino acid sequence (one letter code) of cross linked peptide for USP4 ( purple ) and AKAP18 CD ( gold ). The position of modified lysine’s ( red ) is denoted. Molecular weight markers are indicated on all gels and blots. G , molecular model using PDB coordinates for USP4 (3JYU, purple ) and AKAP18 CD domain (2VFK, gold ) to simulate the docking of both proteins. Positions of cross-linked lysines ( red ) are indicated. AKAP18, A-kinase anchoring protein 18; AMC, 7-amido-4-methylcoumarin; PDB, protein data bank; PKA, protein kinase A; RFU, relative fluorescence unit; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Western Blot, Molecular Weight, Activity Assay, Ub-AMC Assay, Staining, Purification, Incubation, Sequencing, Modification, Fluorescence, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: USP4 is phosphorylated by AKAP18 anchored PKA. A , autoradiograph detecting ( top ) 32 P phosphate incorporation into USP4 immune complexes. Immunoblots confirmed the presence of USP4 ( mid ) and AKAP18 ( bottom ). Experiments were performed without agonist (lane 1) or in the presence of cAMP (lanes 2 and 3), and the peptide kinase inhibitor (PKI). B , GFP tagged AKAP18γ and V5 tagged USP4 were expressed individually or together (indicated below each column) in HEK293 cells. Activity measurements by AMC assay (RFU) of untreated ( gray ) and 10 μm cAMP stimulated ( charcoal ) cells are presented. Deubiquitinase activity above background ( green ) is indicated. Data from four experiments are presented. C , space filling model of USP4 (3JYU, purple ) showing the active site. Serine 829 ( red ) is indicated. (Inset; top ) higher magnification of active site region. Serine 829 ( red ) is indicated. (Inset; bottom ) Conserved consensus PKA motif between residues 821 to 834 of USP4. Serine 829 ( red ) is indicated. Amino acids indicated using one letter code ( D and E ) Characterization of phospho-USP4-Ser 829 antibodies. D , phospho (peptide 1) and nonphospho (peptide 2) analogs of the USP4 821 to 834 sequence were generated by Spot array synthesis. Affinity purified pSer 829 antibodies were evaluated by immunoblot. UV detection of Trp 834 shows amounts of each immobilized peptide. E , maximal cAMP stimulation enhances detection of pSer 829 on USP4. Immunoblot of ( top ) total USP4, ( mid ) pSer 829 and ( bottom ) RII subunit of PKA. Analyses of lysates from unstimulated HCM (lanes 1 and 2) or cells treated with Forskolin/IBMX (10 μM, lanes 3 and 4) to maximize the cAMP response. F , shRNA mediated gene silencing of AKAP18γ and AKAP18δ was performed in HCM cells (lanes 2 and 4). Control cells were treated with scrambled RNA (lanes 1 and 3). Isoproterenol (10 μM) was used as a physiological agonist of the cAMP signaling. Immunoblot detection of ( top ) pSer 829 , ( upper-mid ) total USP4, ( lower mid ) SERCA2 and ( bottom ) AKAP18. Molecular weight markers are indicated for all autoradiographs and immunoblots. G – J , USP4 pSer 829 antisera detects phosphorylation in situ . G and H , immunofluorescent detection of SERCA2 ( cyan ), total USP4 ( green ), and pSer 829 ( magenta ) in paraformaldehyde fixed adult mouse adult cardiomyocytes. Cells treated with ( G ) beta-blocker drug propranolol and ( H ) β-agonist Isoproterenol. The scale bars (20 μm) are indicated. I and J , paraffin embedded tissue section of human heart was subjected to immunofluorescent analyses. Detection of pSer 829 USP4 in ( I ) Normal heart and ( J ) seven day post myocardial infraction. Scale bars (15 μm) are indicated. AKAP18, A-kinase anchoring protein 18; AMC, 7-amido-4-methylcoumarin; HCM, human cardiomyocyte; PKA, protein kinase A; RFU, relative fluorescence unit; SERCA2, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Autoradiography, Western Blot, Activity Assay, Ub-AMC Assay, Sequencing, Generated, Affinity Purification, shRNA, Control, Molecular Weight, Phospho-proteomics, In Situ, Fluorescence, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Long AKAP18 isoforms anchor ubiquitin specific proteinases and coordinate calcium reuptake at the sarcoplasmic reticulum

doi: 10.1016/j.jbc.2025.110317

Figure Lengend Snippet: AKAP18 coordinates aspects of PKA modulation of SERCA2. A , diagram of an AKAP18 signaling island at the sarcoplasmic reticulum. PKA ( green ), SERCA2 ( brown ), phospholamban ( teal ), and USP4 ( magenta ) are indicated. Phosphodiesterase 3 (PDE3, purple ) terminates cAMP signals. Arrows indicate sites of anchored PKA phosphorylation. B , mouse adult cardiomyocytes loaded with Fluo-4 calcium indicator dye were paced at different frequencies (1–4 Hz) with field stimulation. Representative images of ( left ) low and ( right ) high calcium transients in pulsing cells. Scale bars (5 μm) are indicated. C , time course (sec) of calcium transient florescence intensities (494/506 nm) for control ( purple ) and BLU2864-treated ( green ) cardiomyocytes. D – F , amalgamated data (>5 cells per animal n = 3) showing changes in control ( green ) and BLU2864 treated cardiomyocytes. Graphs showing changes in ( D ) peak amplitude (calcium transient relative to resting value); ( E ) rise velocity (rate of the increase of calcium in the cytosol) and ( F ) decay tau (kinetics of calcium clearance) are presented. Numbers of individual cells shown measured below each column. G and H , characterization of AKAP18 −/− mice. G , RII overlay is a modified Western blot procedure used for the detection of AKAPs. Heart lysates (lanes 1 and 2) and AKAP18 immune complexes (lanes 3 & 4) obtained from WT and AKAP18 −/− mice (indicated above each lane) were probed for AKAPs. Molecular weight markers are indicated. H , immunofluorescence detection of AKAP18 in cardiac tissue sections. Representative images from ( left ) WT and ( right ) AKAP18 −/− mice are included. Quantification of fluorescent signal by Fiji/ImageJ (au/100 μm 2 ) for WT ( cyan ) and AKAP18 −/− tissue ( gray ). Number of cells used in each analysis are indicated. I , representative images maximal calcium transients of WT ( left ) and AKAP18 −/− ( right ) in cells. J – L , amalgamated data (numbers of cells shown below each column; n = 5 animals) showing phenotypic differences between WT ( purple ) and AKAP18 −/− ( green ) cardiomyocytes. Graphs showing changes in ( J ) peak amplitude (calcium transient relative to resting value); ( K ) rise velocity (rate of the increase of calcium in the cytosol) and ( L ) decay tau (kinetics of calcium clearance) are presented. All statistical measurements are presented as means ± SEM. AKAP18, A-kinase anchoring protein 18; PKA, protein kinase A; SERCA2a, sarcoplasmic reticulum through the Ca 2+ ATPase 2a; USP, ubiquitin-specific proteinase.

Article Snippet: 1:500 dilution Rabbit polyclonal USP4 antisera (Novus Biologicals, NBP1-86876) was paired with 1:500 dilution Goat anti-AKAP18 to detect anchoring protein/deubiquitinase puncta.

Techniques: Phospho-proteomics, Control, Modification, Western Blot, Molecular Weight, Immunofluorescence, Ubiquitin Proteomics