Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: a , b , Immunoprecipitation (IP) of p22 phox and SERCA2a from mouse whole heart tissue lysates using specific antibodies and with respective IgG control. Immunoprecipitates were loaded along with a 10% input fraction and immunoblotted (IB) for SERCA2a and p22 phox protein. a , Immunoprecipitation of p22 phox with co-immunoprecipitation of SERCA2a (performed at least three times independently). b , Immunoprecipitation of SERCA2a with co-immunoprecipitation of p22 phox (performed at least three times independently). c , In vitro binding assay. Recombinant SERCA2a (rSERCA2a) and recombinant Flag-tagged p22 phox (rFlag-p22 phox ) were incubated in IP cell lysis buffer and immunoprecipitated. The Flag-tagged p22 phox , along with IgG control and the immunoprecipitated complexes, was washed and immunoblotted for SERCA2a and p22 phox . The blots show binding of recombinant Flag-tagged p22 phox (rFlag-p22 phox ) with rSERCA2a (performed at least three times independently). d , PLA performed in neonatal rat cardiomyocytes. Cardiomyocytes were incubated with antibody diluent alone, rabbit anti-p22 phox antibody alone, mouse anti-SERCA2a alone, and anti-p22 phox and anti-SERCA2a antibodies for 2 h at 37 °C. Five random images per well were acquired using DAPI (blue nuclei) and Cy5 filters (red Cy5 PLA fluorescence signals are a result of the secondary antibody-conjugated PLA probe ligation and amplification only in the presence of antibodies against both p22 phox and SERCA2a, indicating close proximity (<40 nm)) (performed at least three times independently). e , Top: in vitro binding of different fragments of recombinant His-tagged SERCA2a (rHis-SERCA2a) with Flag-tagged p22 phox (rFlag-p22 phox ). SERCA2a His-tagged serial truncations were individually incubated with rFlag-p22 phox , then pulled down with anti-Flag M2 beads and immunoblotted with anti-His antibody. The red rectangles indicate the different lengths of recombinant His-tagged SERCA2a (rHis-SERCA2a), seen in the input fraction and the immunoprecipitated lanes that bound to recombinant Flag-tagged p22 phox (rFlag-p22 phox ) (performed at least three times independently). The green rectangles indicate absence of immunoprecipitated bands. Bottom: a schematic representation of the SERCA2a domain structure (three cytoplasmic domains (boxes): the actuator domain (A-domain), the nucleotide binding domain (N-domain) and the phosphorylation domain (P-domain), and transmembrane domains (lines)).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Immunoprecipitation, Control, In Vitro, Binding Assay, Recombinant, Incubation, Lysis, Fluorescence, Ligation, Amplification, Phospho-proteomics

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: a – d , Mouse heart tissues from control and p22 phox cKO mice after sham operation or 1-week TAC were analyzed for mRNA by quantitative PCR and protein expression by immunoblotting. a , Relative SERCA2a mRNA expression in the hearts of control and p22 phox cKO mice (sham: WT—10, p22 phox cKO—11; TAC 1W: WT—8, p22 phox cKO—7). b , Relative PLN mRNA expression in the hearts of control and p22 phox cKO mice (sham: WT—10, p22 phox cKO—11; TAC 1W: WT—8, p22 phox cKO—7). c , Representative immunoblots from the heart LV tissue lysates of control and p22 phox cKO mice after sham and 1-week and 4-week TAC surgery showing changes in SERCA2a protein levels. d , Relative SERCA2a protein expression expressed as the ratio of SERCA2a to GAPDH (sham: WT—8, p22 phox cKO—8; TAC 1W: WT—8, p22 phox cKO—8; TAC 4W: WT—8, p22 phox cKO—8). e , Relative SERCA2a mRNA expression analyzed by quantitative PCR in rat neonatal cardiomyocytes that were transfected with control siRNA or p22 phox siRNA ( n = 9). f , Representative immunoblots of SERCA2a from rat neonatal cardiomyocyte lysates that were transfected with control siRNA or p22 phox siRNA. g , Relative SERCA2a protein levels after normalization to GAPDH ( n = 7). h , Representative immunoblots of T-PLN and phosphorylated (serine 16/17)-PLN (P-PLN (Ser 16/17) from the heart LV tissue lysates of control and p22 phox cKO mice under the sham condition. i , Relative phosphorylated to total phospholamban levels in the heart tissue lysates of control and p22 phox cKO mice under the sham condition ( n = 6). j , The SR fraction was isolated from control and p22 phox cKO (homozygous knockout) mouse hearts, and the specific SERCA2a ATPase activity of the fraction was assessed and normalized to the SERCa2a level in the SR fraction. The graph shows relative ATPase activity (normalized to SERCA2a levels) in the control and p22 phox cKO SR fraction ( n = 8). All bar graphs represent the mean ± s.e. Statistical significance was determined using one-way ANOVA with Tukey test ( a , b and d ) and unpaired Student’s t- test (two tailed) ( e , g , i and j ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Isolation, Knock-Out, Activity Assay, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a – g ) Control and p22 phox cKO mice were injected with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a under cTnT promoter one week prior to sham or TAC surgery. Post-surgery the mice were sacrificed at 4-week time point and the following analyses were conducted ( a ) Representative immunoblots showing SERCA2a levels with GAPDH as loading control in control and p22 phox cKO mice under sham group with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a. ( b ) Relative SERCA2a to GAPDH ratio in (A) (n = 6). ( c ) Representative immunoblots showing SERCA2a levels with GAPDH as loading control in p22 phox cKO mice under sham and TAC (4 W) condition with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a. ( d ) Quantification of SERCA2a to GAPDH ratio in (C) (n = 6). ( e ) Left ventricle weight to tibia length ratio (LVW/TL ratio) in control and p22 phox cKO mice under sham and TAC(4 W) condition with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a (n = 6). ( f ) Lung congestion measured as lung weight to tibia length ratio (Lung W/TL ratio) in control and p22 phox cKO mice under sham and TAC(4 W) condition with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a (n = 6). ( g ) Left ventricle ejection fraction % (LVEF%) measured by echocardiography in control and p22 phox cKO mice under sham and TAC(4 W) condition with adeno-associated virus 9 (AAV9) that harbors either GFP or SERCA2a (n = 6). All bar graphs are represented as Mean ± SE. The statistical significance was determined with 1-way ANOVA with Tukey test (B, D and F-G) and 1-way ANOVA with Šídák’s multiple comparisons test ( e ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Control, Injection, Virus, Western Blot

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: a , Mouse heart tissues from control and p22 phox cKO mice after the sham operation or 4-week TAC were analyzed for dityrosine levels (a marker of oxidative stress on tyrosine residues of proteins) by immunoblotting. The figure shows a representative immunoblot for dityrosine levels with GAPDH as loading control. b , Quantification of dityrosine protein levels as the ratio of relative dityrosine to GAPDH ( n = 6). c , Mouse heart tissues from control and p22 phox cKO mice after the sham operation or 1-week TAC were analyzed for levels of H 2 O 2 released using Amplex red reagent by comparison against known H 2 O 2 standards. The graph shows H 2 O 2 levels in the tissue normalized to the weight of the tissue in mg ( n = 6). d , Rat neonatal cardiomyocytes were transduced with adenoviruses expressing ER-HyPer (ER-targeting ratiometric fluorescence sensor protein that detects H 2 O 2 levels) and co-transfected with control siRNA or p22 phox siRNA. The images shown are representative of the fluorescence ratiometric (excitation peak at 500 nm to excitation peak at 420 nm) images from cells with control siRNA or p22 phox siRNA (the color scale indicates low to high ROS levels) (performed at least three times independently). e , Quantification of the number of high ratio cells to total cells per field from control siRNA or p22 phox siRNA-treated cells, expressed as a percentage ( n = 5). f , Left ventricular homogenates from control and p22 phox cKO mice were incubated with BIAM and pulled down with avidin resin. The pull-down fractions and whole cell lysates (WCL) were immunoblotted for SERCA2a. The figure shows a representative immunoblot of SERCA2a labeled with BIAM from control and p22 phox cKO mouse LV lysates. g , Quantification of BIAM–SERCA2a normalized by total SERCA2a in control and p22 phox cKO mouse LV lysates (data from six mice per group). h , The overlay extract ion chromatograms of peptide SM ox SVYC 498-biotin TPNKPSR in control and KO. The PEO-iodoacetyl-LC-biotin modification on cysteine represents the reduced form of cysteine. The dramatic decrease in the amount of the reduced form of cysteine at C498 in the KO indicates that the KO has more oxidized C498. i – m , Neonatal rat ventricular myocytes (NRVMs) were transduced with Ad–Flag–SERCA2a WT or Ad–Flag–SERCA2a-C498S mutant in the presence or absence of p22 phox siRNA. i , Representative immunoblots of BIAM-labeled Flag-SERCA2a obtained from NRVMs that were transfected with Ad–Flag–SERCA2a WT or Ad–Flag–SERCA2a-C498S mutant in the presence or absence of p22 phox siRNA. SE, short exposure; LE, long exposure. j , Quantification analyses of BIAM–Flag–SERCA2a normalized by total Flag–SERCA2a in l ( n = 6). k , Representative immunoblots of Flag–SERCA2a in rat neonatal cardiomyocytes transfected with Ad–Flag–SERCA2a WT or Ad–Flag–SERCA2a-C498S mutant in the presence or absence of p22 phox siRNA. l , Quantification of the Flag–SERCA2a WT protein expression levels in k ( n = 12). m , Quantification of the Flag–SERCA2a-C498S mutant protein expression in k ( n = 12). All bar graphs represent the mean ± s.e. Statistical significance was determined using one-way ANOVA with Tukey test ( b , c and j ) and unpaired Student’s t- test (two tailed) ( e , g , l and m ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Control, Marker, Western Blot, Comparison, Transduction, Expressing, Fluorescence, Transfection, Incubation, Avidin-Biotin Assay, Labeling, Modification, Mutagenesis, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a , b ) Neonatal rat cardiomyocytes were transfected with control siRNA, p22 phox siRNA, Nox4 siRNA or p22 phox siRNA + Nox4 siRNA and transduced with adenovirus harboring ER-localized HyPer. After 48 h incubation, the cells were imaged live under a confocal microscope and the ratiometric image indicating ER H 2 O 2 levels was generated and quantified. ( a ) Representative ratiometric image of ER-HyPer fluorescence. Color scale indicates low to high ratio grading. Scale bar = 100 µm. ( b ) Quantification of ER-HyPer high ratio cells in ( a ) ( n = 5). ( c – e ) Neonatal rat cardiomyocytes were transfected with control siRNA, p22 phox siRNA, Nox4 siRNA or p22 phox siRNA + Nox4 siRNA. Nox4 was overexpressed as a positive control to identify the specific protein band of Nox4 . ( c ) Representative immunoblot showing protein levels of SERCA2a, Nox4 and p22 phox with GAPDH as loading control and Nox OE indicating the specific Nox4 protein band. ( d , e ) Quantification of Nox4 and SERCA2a protein levels normalized to GAPDH levels in ( c ) ( n = 9). All bar graphs are represented as mean + SE. The statistical significance was determined by 1-way ANOVA with Tukey test ( b , d and e ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Transfection, Control, Transduction, Incubation, Microscopy, Generated, Fluorescence, Positive Control, Western Blot

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a , b ) Neonatal rat cardiomyocytes were transfected with the indicated siRNAs and after 48 h the cell lysates were analyzed by Western blotting. ( a ) Representative immunoblot showing protein levels of Nox2 under control siRNA- and p22 phox siRNA-treated conditions. ( b ) Quantification of Nox2 protein levels expressed as the ratio to GAPDH levels in ( a ) ( n = 6). ( c , d ) Neonatal rat cardiomyocytes were transfected with the indicated siRNAs and transduced with adenovirus harboring mitochondria-localized HyPer. After 48 h incubation, the cells were imaged live under a confocal microscope and the ratiometric image indicating mitochondrial H 2 O 2 levels was generated and quantified ( c ). Representative ratiometric image of Mito-HyPer fluorescence. Color scale indicates low to high ratio grading. Scale bar = 100 µm. ( d ) Quantification of Mito-HyPer high ratio cells in ( c ) ( n = 5). ( e , f ) Neonatal rat cardiomyocytes were transfected with the indicated control siRNAs or SERCA2a siRNA and transduced with adenovirus harboring ER-localized HyPer. After 48 h incubation, the cells were imaged live under a confocal microscope and the ratiometric image indicating ER H 2 O 2 levels was generated and quantified. ( e ) Representative ratiometric image of ER-HyPer fluorescence. Color scale indicates low to high ratio grading. Scale bar = 100 µm. ( f ) Quantification of ER-HyPer high ratio cells in ( e ) ( n = 5). All data in bar graphs are represented as mean + SE. The statistical significance was determined with unpaired Student’s t test (2 tailed) ( b , d and f ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Transfection, Western Blot, Control, Transduction, Incubation, Microscopy, Generated, Fluorescence

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a – d ) Neonatal rat ventricular myocytes (NRVM) were transduced with adenovirus harboring either WT SERCA2a-FLAG or SERCA2a (C498S)-FLAG and adenovirus harboring LacZ or p22 phox shRNA. The stability of SERCA2a was evaluated by pre-incubation (for 1 h) with or without epoxomicin (Epo) (50 µM) (20 s proteasome inhibitor). After treatment with cycloheximide (CHX) (15 µg/ml), cells were collected at 0, 3, 6 and 9 h and analyzed for protein levels of either WT SERCA2a-FLAG or SERCA2a (C498S)-FLAG. ( a ) Representative immunoblots of WT SERCA2a-FLAG post-CHX treatment with or without epoxomicin pre-incubation. ( b ) Representative immunoblots of SERCA2a(C498S)-FLAG post-CHX treatment with or without epoxomicin pre-incubation. ( c ) Quantification of percentage of WT SERCA2a-FLAG plotted for each time point. Each group is indicated by color codes as shown in the graphs. Each dot represents mean ± SE for each time point and connecting lines indicate trend. P values indicated in red fonts compare (Ad: Lac Z vs Ad: p22 phox shRNA groups). P values indicated in black fonts compare (Ad: p22 phox shRNA vs Ad: p22 phox shRNA + Epo groups) at the respective time points. The P values were compared within each timepoint by unpaired Student’s t test (2 tailed) ( n = 5). ( d ) Quantification of percentage of SERCA2a (C498S)-FLAG plotted for each time point. Each group is indicated by color codes as shown in the graphs. Each dot represents mean ± SE for each time point and connecting lines indicate trend. The P values were compared within each timepoint by unpaired Student’s t test (2 tailed) and are not significant (ns) ( n = 5). ( e , f ) NRVMs were transfected with control siRNA or p22 phox siRNA along with PSMA3 siRNA in combinations as indicated in the figure for 48 h, and cell lysates were analyzed by immunoblotting for protein levels of SERCA2a. ( e ) Representative immunoblots of SERCA2a with α-tubulin as loading control from cell lysates after indicated siRNA treatment. ( f ) Quantification of relative SERCA2a levels in ( e ) ( n = 6). The bar graph is represented as mean ± SE. The statistical significance was determined with 1-way ANOVA with Tukey test.

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Transduction, shRNA, Incubation, Western Blot, Transfection, Control

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a ) NRVMs were transduced with adenovirus expressing HA-SERCA2a (334-666) fragment (minigene) in increasing amounts as indicated in plaque forming unit (pfu), and cells were collected and analyzed by immunoblotting for SERCA2a protein levels. The figure shows a representative immunoblot of SERCA2a levels with α-tubulin as loading control. The expression of HA-SERCA2a (334-666) fragment is also validated. ( b ) Quantification of SERCA2a levels in ( a ) ( n = 7). ( c , d ) NRVMs were transduced with Ad-HA-SERCA2a (334-666) (minigene) or Ad-LacZ (control) and the cell lysates were immunoprecipitated to study interactions. ( c ) p22 phox was detected in the anti-HA immunoprecipitate but not in the control immunoprecipitate in Ad-HA-SERCA2a (334-666) (minigene) transduced NRVMs (at least performed three times independently). ( d ) Immunoprecipitation was performed with either anti-SERCA2a or control antibody. The interaction between SERCA2a and p22 phox was attenuated in the presence of HA-SERCA2a (334-666) (minigene) (at least performed three times independently). ( e , f ) NRVMs were transduced with Ad-HA-SERCA2a (334-666) (minigene) or Ad-LacZ (control) and treated with DMSO (vehicle) or MG132 (proteasome inhibitor) and the cell lysates were analyzed for SERCA2a protein levels by immunoblotting. ( e ) Representative SERCA2a immunoblot with GAPDH as loading control shows SERCA2a protein level was stabilized in the presence of MG132 in both the presence and absence of Ad-HA-SERCA2a (334-666) minigene. ( f ) Quantification of relative SERCA2a protein levels in ( e ) ( n = 6). ( g ) NRVMs were transduced with Ad-HA-SERCA2a (334-666) (minigene) or Ad-LacZ (control) or treated with H 2 O 2 (positive control for oxidation) (vehicle). Cells were collected after treatment with MG132 (proteasome inhibitor) for 6 h. Cell lysates were incubated with biotinylated iodoacetamide (BIAM) and pulled down with avidin resin. The pulldown and input fractions were immunoblotted for SERCA2a. SERCA2a oxidation was promoted (indicated by decreases in BIAM pull-down) in the presence of SERCA2a (334-666) and H 2 O 2 . ( h ) Quantification of BIAM-SERCA2a to total SERCA2a ratio in ( g ) ( n = 6). ( i , j ) AAV9-cTNT-FLAG-mSERCA2a (333-666) or AAV9-cTNT-eGFP was injected into wild type (WT) mice and the level of SERCA2a in the heart was evaluated by immunoblotting. ( i ) Representative immunoblots of SERCA2a with α-tubulin as loading control. FLAG-mSERCA2a (333-666) expression is validated. ( j ) Quantification of SERCA2a to GAPDH ratio in ( i ) ( n = 6). All bar graphs are represented as mean ± SE. The statistical significance was determined with 1-way ANOVA with Tukey test ( b , f ), 1-way ANOVA with Dunnett’s multiple comparisons test ( h ) and unpaired Student’s t test (2 tailed) ( j ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Transduction, Expressing, Western Blot, Control, Immunoprecipitation, Positive Control, Incubation, Avidin-Biotin Assay, Injection

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: a , NRVMs were transfected with control siRNA or p22 phox siRNA for 48 h and treated with MG132 for 3 h. The cell lysates were immunoprecipitated with either SERCA2a antibody or IgG control antibody and checked by immunoblotting for interaction with Smurf1 or Hrd1 E3 ubiquitin ligases. The immunoblots show enhanced binding of both Smurf1 and Hrd1 E3 ubiquitin ligases with SERCA2a in the absence of p22 phox . Epoxomicin treatment at baseline also showed SERCA2a interaction with Hrd1 and Smurf1 (performed at least three times independently). b – e , NRVMs were transfected with control siRNA or p22 phox siRNA together with Smurf1 or Hrd1 siRNA as indicated for 48 h. Cell lysates were collected and analyzed for protein levels of SERCA2a by immunoblotting. b , Representative immunoblots of SERCA2a with GAPDH as loading control. Knockdown of Smurf1 and p22 phox is also validated. c , Relative SERCA2a protein levels in b ( n = 6). d , Representative immunoblots of SERCA2a with GAPDH as loading control. Knockdown of Hrd1 and p22 phox is also validated. e , Relative SERCA2a protein levels in c ( n = 6). f – h , NRVMs were transfected with control siRNA or p22 phox siRNA for 48 h and stained by immunofluorescence. Images were acquired by confocal microscopy. The blue color indicates DAPI (nucleus), green is SERCA2a, red is Hrd1 and yellow is the colocalization channel. f , Representative all-channel confocal microscopy images showing colocalization of SERCA2a and Hrd1. g , Three-dimensional (3D) fluorescence reconstructed image using IMARIS software after confocal microscopic image acquisition with z -stacks. h , The quantification of colocalization was calculated as the ratio of the volume of colocalization to the volume of SERCA2a in each cell following IMARIS software analysis ( n = 6). i , Representative immunofluorescence images from control siRNA or p22 phox siRNA-treated NRVMs stained with DAPI (white: pseudo color), SERCA2a (red), Hrd1 (blue) and PSMA3 (green). Individual channels are shown separately, and the merge channel is shown enlarged. The purple arrows indicate colocalization of SERCA2a with Hrd1, and the white arrows indicate triple colocalization of SERCA2a, Hrd1 and PSMA3. j , The quantification of triple colocalization was calculated as the ratio of the volume of triple colocalization to the volume of SERCA2a in each cell following IMARIS software analysis ( n = 5). All bar graphs represent the mean ± s.e. Statistical significance was determined using one-way ANOVA with Tukey test ( c and e ) and unpaired Student’s t -test (two tailed) ( h and j ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Binding Assay, Knockdown, Staining, Immunofluorescence, Confocal Microscopy, Fluorescence, Software, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: Control (wild type +/+ MHC cre + or p22 phox flox/flox/+ cre−), p22 phox cKO (flox/flox MHC cre+), SERCA2a-C498S heterozygous knock-in (C498S het KI) and p22 phox cKO with SERCA2a-C498S heterozygous knock-in were subjected to sham surgery or transverse aortic constriction (TAC 3W). The mice were then analyzed for hypertrophic response and cardiac function by echocardiography. The heart LV tissues were collected for the indicated analyses. a , Representative echocardiographs from control, p22 phox cKO, SERCA2a-C498S heterozygous knock-in and p22 phox cKO with SERCA2a-C498S heterozygous knock-in mice after sham surgery or TAC 3W. b , LVEF (%) (sham: n = 6, 7, 9, 7; TAC: n = 7, 6, 7, 7). c , LVW/TL ratio (sham: n = 7, 5, 8, 5; TAC: n = 5, 7, 7, 7). d , Lung W/TL ratio ( n = 7). e , The heart LV tissue of the mice that were subjected to sham or TAC 3W was analyzed by western blotting for protein levels of SERCA2a, with GAPDH as loading control. The figure shows representative immunoblots of SERCA2a with GAPDH as loading control from control and p22 phox cKO mice with wild type or C498S mutant SERCA2a. f , Quantification of SERCA2a levels with respect to GAPDH in e ( n = 6). g , h , Cardiomyocyte contractility was assessed in isolated cardiomyocytes from the indicated groups under NT solution and isoproterenol (1 µM) (Iso)-stimulated conditions. For each group, four mice and the specified number of cells per mouse were recorded and summarized. g , Representative cell shortening calculated as a percentage with electrical stimulation (3 s duration) indicated with arrows. h , Quantification graph of the percentage cardiomyocyte shortening in g (from left to right, n = 6, 6, 4, 6; n = 5, 5, 4, 6; n = 6, 4, 5, 11; n = 9, 7, 6, 5; n = 6, 6, 3, 3; n = 5, 6, 3, 4; n = 7, 8, 8, 5; n = 7, 7, 8, 8). The data points from cells of each mouse are represented by colored dots (black, blue, brown and black empty). All bar graphs represent the mean ± s.e. Statistical significance was determined with one-way ANOVA with Tukey test ( b – d and f ). Statistical analysis was conducted using the nested t- test (two tailed) ( h ) and hierarchical clustering analysis and the dendrogram is provided in the source data.

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Control, Knock-In, Western Blot, Mutagenesis, Isolation, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a – d ) Ca 2+ transient in WT, p22 phox cKO. SERCA2a C498S homozygous KI and p22 phox cKO + SERCA2a C498S homozygous KI cardiomyocytes at baseline and with isoproterenol (1 µm) stimulation. Adult cardiomyocytes were isolated from respective mice using the Langendorff perfusion method. The cardiomyocytes were loaded with Fluo-4 AM. Ca 2+ fluorescence intensity was recorded as the ratio of the fluorescence (F) to the basal diastolic fluorescence (F0). ( a ) Representative twitch Ca 2+ transients under normal Tyrodes (NT) and with isoproterenol (1 µm) (Iso), measured as the height of the peak of Ca 2+ fluorescence intensity recorded as the ratio F/F0. ( b , c ) Summarized data represented as mean ± SE for ( b ) twitch Ca 2+ transient amplitude (from left to right n = 5,5,5; n = 5,5,5; n = 4,4,7; n = 4,4,7; n = 5,9,10, n = 5,9,13; n = 6,4,3; n = 6,4,3), ( c ) decay constant (Tau) in ( a ) (from left to right n = 5,6,5; n = 5,6,5; n = 5,5,5; n = 5,5,5; n = 5,5,5; n = 5,5,5; n = 5,5,5; n = 5,5,5). 3 mice per condition and indicated number of cells per mice were recorded and summarized. The data points from cells of each mouse are represented with distinct color dots (black, blue and black empty). The statistical analysis was conducted using the nested t test (2 tailed) ( b and c ) and hierarchical clustering analysis was performed and the dendrogram is provided in the source data file.

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Isolation, Fluorescence

Journal: Nature Cardiovascular Research

Article Title: p22 phox prevents the oxidation of SERCA2a and stabilizes it in the heart

doi: 10.1038/s44161-025-00699-x

Figure Lengend Snippet: ( a – f ) Human heart samples obtained from non-heart failure (non-HF) (donors) and heart failure (HF) patients (recipients) were analyzed by immunofluorescence and immunoblotting. ( a ) Representative immunofluorescence staining of non-heart failure (non-HF) (donors) and heart failure (HF) patients with SERCA2a antibody (Red fluorescence) and blue DAPI (nucleus). Scale bar = 200 µm. ( b ) Quantification of the relative SERCA2a fluorescence intensity in non-heart failure (non-HF, n = 7) (donors) and heart failure (HF, n = 6) patients. ( c ) Immunoblot of lysates from non-heart failure (non-HF) (donors, n = 7) and heart failure (HF) patients (recipients, n = 7) with dilated cardiomyopathy (DCM, n = 4) or ischemic cardiomyopathy (ICM, n = 3) showing SERCA2a, Hrd1 and Smurf1 levels, with GAPDH as loading control. ( d – f ) Quantification of indicated protein levels as the ratio to GAPDH, compared between donors and recipients. All bar graphs are represented as mean + SE. The statistical significance was determined with unpaired Student’s t test (2 tailed) ( b ) and the Mann Whitney test (2 tailed) ( d – f ).

Article Snippet: Recombinant SERCA2a protein was purchased from Lifespan Biosciences ( G27566 ).

Techniques: Immunofluorescence, Western Blot, Staining, Fluorescence, Control, MANN-WHITNEY

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Gastrodin Pretreatment Impact on Sarcoplasmic Reticulum Calcium Transport ATPase (SERCA) and Calcium Phosphate (PLB) Expression in Rats with Myocardial Ischemia Reperfusion

doi: 10.12659/MSM.896835

Figure Lengend Snippet: Gastrodin pretreatment impact on SERCA 2a, PLB, and pSer16-PLB protein expression in myocardial tissue. A, control; B, model group; C, D, E, gastrodin pretreatment group. # P<0.05, compared with group A; * P<0.05, compared with group B; & P<0.05, compared with group C; $ P<0.05, compared with group D.

Article Snippet: SERCA2a, PLB, and pSer16-PLB protein antibodies were provided by ABR, Santa Cruz Biotechnology, USA.

Techniques: Expressing, Control