Journal: bioRxiv

Article Title: Quantitative Proteomics Links Mitochondrial Dysfunction to Metabolic Changes and Epithelial Differentiation Defects in Hyperoxia-Exposed Neonatal Airway Cells

doi: 10.1101/2025.06.15.659571

Figure Lengend Snippet: (A) Experimental timeline for neonatal tracheal aspirate collection to ALI culture of nTAECs with hyperoxia exposure. (B) Normal airway mucociliary differentiation from basal cells. (C) Quantitative PCR analysis of epithelial cell-specific markers for basal (KRT5), club (SCGB1A1), ciliated (FOXJ1), and goblet (MUC5AC) cells on ALI day 14 compared to ALI day 0. Relative gene expression was normalized to GAPDH (endogenous control) and plotted as fold change over ALI day 0. Data presented as mean (n = 4 donor samples. Solid black circles represent males; open circle represents female). Error bars indicate SEM. Statistical analysis was performed using Mann-Whitney U test. (D-E) Immunofluorescent staining with representative images of airway progenitor (P63⍺, red) performed on ALI day 0 and 14, and club (SCGB1A1, red), goblet (MUC5AC, green) ciliated (ACTUB, green) cells were performed on ALI day 14. Nuclei are counterstained blue with DAPI. Scale bar = 50 µm.

Article Snippet: Samples were then incubated for 2 hours at room temperature with primary antibodies targeting key epithelial cell markers: P63α (basal cells; 1:100, Cat# 13109S, Cell Signaling Technology), Secretoglobin 1A1 or SCGB1A1 (club cells; 1:200, Cat# RD181022220-01, BioVendor LLC), Mucin-5AC or MUC5AC (goblet cells; 1:100, Cat# MA5-12178, Thermo Scientific), and acetylated tubulin or ACTUB (ciliated cells; 1:200, Cat# T7451, Sigma-Aldrich).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Control, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: Quantitative Proteomics Links Mitochondrial Dysfunction to Metabolic Changes and Epithelial Differentiation Defects in Hyperoxia-Exposed Neonatal Airway Cells

doi: 10.1101/2025.06.15.659571

Figure Lengend Snippet: (A) Immunofluorescent staining of airway progenitor (P63⍺, red), ciliated (ACTUB, green), club (SCGB1A1, red) and goblet (MUC5AC, green) cells were performed on ALI 14 following room air or hyperoxia exposure. Nuclei are counterstained blue with DAPI. Each data points represent the ratio of positive cells for each donor (n = 6 donor sample. Solid black circles represent males; open circle represent female) normalized to room air control. Statistical analysis was performed Mann-Whitney U test. Scale bar = 50 µm. (B) TEER measurement on ALI day 14 comparing room air and hyperoxia exposed nTAECs do not show any significant difference. Data points represent average TEER values for each donor (n=5 donors, 3-4 wells per donor. Solid black circles represent males; open circle represent female). Statistical analysis was performed Mann-Whitney U test.

Article Snippet: Samples were then incubated for 2 hours at room temperature with primary antibodies targeting key epithelial cell markers: P63α (basal cells; 1:100, Cat# 13109S, Cell Signaling Technology), Secretoglobin 1A1 or SCGB1A1 (club cells; 1:200, Cat# RD181022220-01, BioVendor LLC), Mucin-5AC or MUC5AC (goblet cells; 1:100, Cat# MA5-12178, Thermo Scientific), and acetylated tubulin or ACTUB (ciliated cells; 1:200, Cat# T7451, Sigma-Aldrich).

Techniques: Staining, Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) The interaction between SAV1 and PPARγ was enhanced by MST2. 293 cells were transfected with HA-tagged wild-type (WT) or inactive (KD) MST2, Myc-SAV1 and/or Flag-PPARγ, and the interaction between PPARγ and SAV1 or MST2 was detected by immunoprecipitation with an anti-Flag antibody followed by immunoblotting with an anti-Myc or an anti-HA antibody. (B) Direct binding of PPARγ and SAV1 was confirmed by in vitro pull-down assay using recombinant hexahistidine-tagged SAV1 protein and PPARγ and MST2 proteins that were overexpressed in 293 cells. (C) MST1 also enhanced the interaction between SAV1 and PPARγ. The experiment was performed as described in (A). (D) The interaction between endogenous SAV1 and PPARγ was detected in 3T3-L1 adipocytes by immunoprecipitation with an anti-PPARγ antibody followed by immunoblotting with an anti-SAV1 antibody or inversely, immunoprecipitation with an anti-SAV1 antibody followed by immunoblotting with an anti-PPARγ antibody.

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Transfection, Immunoprecipitation, Western Blot, Binding Assay, In Vitro, Pull Down Assay, Recombinant

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) Myc-tagged SAV-1 deletion mutants were co-expressed with Flag-tagged PPARγ and HA-tagged MST2 in 293 cells. After 48 h, cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with an anti-Myc antibody (upper panel). As a control, whole cell lysates were immunoblotted with the indicated antibodies (lower panels). (B) As above, the interactions of Flag-tagged PPARγ deletion mutants with Myc-tagged SAV-1 were analyzed. (C) Mutation of PPYY to PPYA in PPARγ decreases its interaction with SAV1. D. Schematic of the domain structure of SAV1 and PPARγ2.

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Immunoprecipitation, Mutagenesis

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) Increasing amounts (0.5, 1, 3 µg) of Myc-SAV1 were co-expressed with Flag-PPARγ and/or HA-MST2 in 293 cells. At 48 h after transfection, cell lysates were immunoblotted with the indicated antibodies. (B) Flag-tagged PPARγ or PPARγ(128–505) was co-expressed with HA-MST2, HA-MST2-KD (inactive mutant), Myc-SAV1, and/or Myc-SAV1(1–201) in 293 cells as indicated. Cell lysates were prepared at various times after treatment with 40 µg/mL cycloheximide and then immunoblotted with an anti-Flag antibody. The PPARγ protein expression was quantified and the half-life of PPARγ protein was calculated. Expression of GAPDH was detected as a loading control.

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Transfection, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) The expression of SAV1 and MST2 increased during the differentiation of 3T3-L1 cells. Expression of the indicated proteins and phosphorylation of MST2 were analyzed with antibodies in the lysates of 3T3-L1 cells prepared at various times after induction of differentiation. (B) The levels of endogenous PPARγ proteins were increased by overexpression of MST2 and/or SAV1 in 293 cells. (C) The levels of endogenous PPARγ proteins were increased by lentivirus-mediated overexpression of MST2 or SAV1 in 3T3-L1 adipocytes. (D) The levels of endogenous PPARγ proteins were decreased by shRNA-mediated knockdown of SAV1 expression in 3T3-L1 adipocytes. (E) The degradation of PPARγ protein was proteasome-dependent. The decrease of PPARγ protein induced by knock-down of SAV1 expression was inhibited by 5 µM lactacystin, a proteasome inhibitor.

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Expressing, Over Expression, shRNA

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) The transactivation activity of PPARγ increased upon co-expression of MST2 and SAV1. U2OS cells were transfected with a reporter gene, aP2-Luc, and pRL-TK along with PPARγ, SAV1 and/or MST2. Luciferase activity was assayed for the cells treated with (black bar) or without (white bar) 20 µM rosiglitazone. The lower panel shows expression of PPARγ and expression of GAPDH was detected as a loading control. (B) Full-length (FL) and deletion mutants of SAV1, SAV1(1–201) and SAV1(1–267) were transfected together with MST2 and PPARγ, and luciferase activity was assayed. (C) The mRNA level of adiponectin, a PPARγ target gene, was increased significantly by co-expression of MST2 and SAV1. Quantitative real-time PCR was performed with cDNA prepared from 3T3-L1 adipocytes transfected with MST2 and SAV1 as indicated. (D) The rosiglitazone-induced activation of PPARγ was inhibited by knockdown of SAV1 expression with siRNA against SAV1. All values are expressed as the mean ± SD (n = 3).

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Real-time Polymerase Chain Reaction, Activation Assay

Journal: PLoS ONE

Article Title: Mammalian Ste20-Like Kinase and SAV1 Promote 3T3-L1 Adipocyte Differentiation by Activation of PPARγ

doi: 10.1371/journal.pone.0030983

Figure Lengend Snippet: (A) Overexpression of MST2 and SAV1 stimulated differentiation of 3T3-L1 adipocytes. 3T3-L1 pre-adipocytes were transfected with PPARγ, SAV1 and/or MST2 expression plasmids and then stimulated with the minimal differentiation cocktail. After 10 d, cells were stained with Oil Red O and photographed using a microscope (200× magnification). (B) Kinase activity was required for the stimulation of differentiation of 3T3-L1 adipocytes by MST2. The experiment was performed as described in (A). (C) For quantification of samples from (A) and (B), Oil Red O stain was extracted and absorbances were measured at 520 nm. The lower panel confirms the expression of transfected proteins by immunoblotting. (D) Knockdown of SAV1 using shRNA inhibited differentiation of 3T3-L1 adipocytes. 3T3-L1 pre-adipocytes were transfected with a SAV1shRNA plasmid or a control plasmid and then stimulated with the full differentiation cocktail. After 10 d, cells were stained with Oil Red O and photographed. (E) For quantification of samples from (D), Oil Red O stain was extracted and absorbances were measured at 520 nm. The lower panel confirms the knockdown of SAV1 by immunoblotting. (F) Knockdown of MST1 or MST2 using siRNA inhibited the differentiation of 3T3-L1 adipocytes. The experiment was performed as described in (D). (G) For quantification of samples from (F), Oil Red O stain was extracted and absorbances were measured at 520 nm. The lower panel confirms the knockdown of MST1 or MST2 expression. All values are expressed as the mean ± SD (n = 3).

Article Snippet: Our antibody against SAV1 raised in rabbit with bacterially expressed human SAV1 protein (AbFrontier, Korea) was also used.

Techniques: Over Expression, Transfection, Expressing, Staining, Microscopy, Activity Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Identification of BBLN in TOF patient heart specimens by nano-LC–ESI–MS/MS analysis of CAMK2D-co-enriched proteins in the 8–14 kDa range. b , AP of CAMK2D from cardiac specimens of TOF patients (AP: CAMK2D) followed by IB detection of enriched CAMK2D (IB: CAMK2D) and co-enriched BBLN (IB: BBLN). The control immunoaffinity matrix (AP: con) did not enrich CAMK2D nor BBLN. The experiment was repeated three times with similar results. c , Characteristics of TOF patients who were included in the study for the immunohistological determination of cardiac BBLN. Age between acyanotic and cyanotic TOF patients was comparable while oxygen saturation (sat.) was significantly different. d , Immunohistological detection of BBLN on cardiac specimens of TOF patients without (left) and with cyanosis (right). Scale bar, 40 μm. Immunohistological detection of BBLN was performed on specimens of 16 acyanotic TOF patients and 16 cyanotic TOF patients (cf. f and g ). e – g , Immunohistological determination of BBLN on cardiac specimens of 16 acyanotic TOF patients – and 16 cyanotic TOF patients – . Counterstaining was performed with hematoxylin (HE). Quantitative BBLN data ( e ) and immunohistological images of cardiac specimens from acyanotic TOF patients ( f ) and cyanotic TOF patients ( g ) are shown. Scale bar, 2 mm. Data are mean ± s.d. ( n = 16 patients per group; unpaired, two-tailed t -test; d.f. 30; c , t = 2.010 and 7.820, P = 0.05354 and 9.99913 × 10 −9 ; e , t = 7.811 and P = 1.02375 × 10 −8 ).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Tandem Mass Spectroscopy, Control, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Immunofluorescence colocalization of BBLN with CAMK2D on a heart specimen of an 8-month-old, male Tg- BBLN mouse (scale bar, 40 μm). The immunofluorescence colocalization was performed with heart specimens of five different Tg- BBLN mice (Tg-1) and five different nontransgenic FVB controls. All immunofluorescence images are shown in Supplementary Fig. . b , Quantitative IB determination of cardiac contents of activated phospho-T287–CAMK2D, inactive phospho-T307–CAMK2D, total CAMK2D and BBLN in 8-month-old, Tg- BBLN mice (Tg-1) and age- and sex-matched nontransgenic FVB control mice. The lower control IB detects ATP5A1. Left: IB images and right: quantitative IB data (mean ± s.d., n = 9 mice per group, 5 males and 4 females; unpaired, two-tailed t -test; d.f. 16, t = 32.92, 8.467, 2.602 and 56.01; P = 3.9625 × 10 −16 , 2.63699 × 10 −7 , 0.01927 and 8.64361 × 10 −20 ). c , d , In vitro data show that recombinant BBLN protein enhanced the autophosphorylation of recombinant CAMK2D (200 nM) and the CAMK2D-mediated substrate phosphorylation of recombinant PDC and BBLN. Representative autoradiography images ( c ) and quantitative data ( d ) of BBLN-enhanced PDC phosphorylation by CAMK2D (50 nM) in vitro, in the presence and absence (w/o) of Ca 2+ +calmodulin (CALM) (mean ± s.d., n = 3 biological replicates, one-way ANOVA and Dunnett’s test; F (9,20) = 38.33; P = 0.9998, 0.3728, <0.0001 and <0.0001 BBLN+Ca 2+ +CALM versus Cont.+Ca 2+ +CALM; ** P = 0.0052 versus Cont.+Ca 2+ +CALM; * P = 0.0182, 0.0321, 0.0113 and 0.0135 versus Cont.+Ca 2+ +CALM). e , Quantitative IB determination of cardiac contents of phospho-T287–CAMK2D, total CAMK2D, phospho-S2813–RYR2 and BBLN (and BBLN–SxxA) in 8-month-old, male Tg- BBLN mice (Tg-2), nontransgenic FVB mice and Tg- BBLN mice (Tg-2) after 4 weeks of lentiviral transduction of miCamk2d (Tg- BBLN +miCamk2d) and Tg- BBLN –SxxA mice. The control IB detects ATP5A1. Quantitative data (left) and IB images (right) (mean ± s.d.; n = 4 male, 8-month-old mice per group, one-way ANOVA and Tukey’s test; F (3,12) = 111.4, 27.29, 62.58 and 52.25; upper left: P = 1.914 × 10 −7 , 5.873 × 10 −9 and 3.424 × 10 −8 ; lower left: P = 0.4303, 0.00001428 and 0.5147; upper right: P = 0.000007374, 8.287 × 10 −8 and 0.000004093; lower right: P = 0.0001016, 0.0933 and 0.001294); f , The left ventricular EF and the LVIDd of 8-month-old, male mice were determined by echocardiography (mean ± s.d., n = 6 mice per group, one-way ANOVA and Tukey’s test; F (3,20) = 54.28 and 5.562; P = 1.19 × 10 −9 , 3.58 × 10 −8 , 6.628 × 10 −8 EF; P = 0.004012, 0.1793 and 0.0449 LVIDd).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Immunofluorescence, Control, Two Tailed Test, In Vitro, Recombinant, Autoradiography, Transduction

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , e Cardiac transcript levels of different Camk2d splice variants were determined by NGS in right ( a ) and left ventricles ( e ) of 3–4-month-old, male non-transgenic FVB mice and transgenic Tg- BBLN mice (Tg-1), and are presented as TPM (transcripts per million). b - d , f-h , Transcript levels of cardiac Camk2a , Camk2b and Camk2g were determined by NGS in right ventricular ( b-d ) and left ventricular heart specimens ( f-h) of non-transgenic FVB mice and transgenic Tg- BBLN mice. Data are mean ± s.d. P-values were determined by the unpaired, two-tailed t-test; just alpha; n = 4 mice per group (df=6; a , t = 2.103, 2.494, 4.952, 1.473; p = 0.0801, 0.0469, 0.0026, 0.1913; b , t = 6.433; p = 0.0007; c , t = 13.39; p = 1.0757e-05; d , t = 1.727; p = 0.1349; e , t = 3.453, 0.5919, 4.256, 1.050; p = 0.0136, 0.5755, 0.0053, 0.3342; f , 5.857; p = 0.0011; g , 4.797; p = 0.0030; h , 0.1269; p = 0.9032).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Transgenic Assay, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Immunoblot analysis detected reduced total cardiac RYR2 protein contents and increased CAMK2D-mediated phosphorylation of RYR2 on serine-2813 in Tg- BBLN mice (Tg-1) compared to non-transgenic FVB controls. Representative immunoblots (left panels), and quantitative immunoblot data (right panels) are shown. b , Cardiac ATP2A2 (SERCA2) levels are decreased in Tg- BBLN (Tg-1) hearts. The lower control blot detects ATP5A1. Representative immunoblots (left panels), and quantitative data (right panels) are shown. Data ( a , b ) are mean ± s.d. (n = 9 mice per group, 5 male, 4 female; age: 8 months). P-values were determined by the unpaired, two-tailed t-test (df=16; a , t = 9.4818, 4.9131; p = 5.7293e-08, p = 0.000156; b , t = 11.09, 0.3828; p = 6.42141e-09, p = 0.7069). c , Transcript levels of Ryr2 (left panel) and Atp2a2 (right panel) were determined by NGS in heart specimens of Tg- BBLN mice (Tg-1) and non-transgenic FVB mice. Data are mean ± s.d. (n = 4 male mice per group; age: 8 months). P-values were determined by the unpaired, two-tailed t-test (df=6; t = 7.4612, p = 0.000299 ( Ryr2 ); t = 33.28, p = 4.8989e-08 ( Atp2a2 )). d , BBLN transcript levels in right ventricular heart specimens of TOF patients with cyanosis were determined by microarray analysis (Affymetrix probe set ID: 204480_s_at) and negatively correlated with RYR2 (214044_at) and ATP2A2 (212361_s_at). Linear regression analysis was performed, and the Pearson correlation coefficient (r) and p-values (left panel, two-tailed, p = 0.0412; right panel, one-tailed, p = 0.0486) were determined (n = 11 TOF patients with cyanosis, 7 females, 4 males; age: 23.2 ± 5 months).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Western Blot, Transgenic Assay, Control, Two Tailed Test, Microarray, One-tailed Test

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Autophosphorylation of CAMK2D on T287 in the absence (Cont.) and presence of BBLN, BBLN-Mut1, BBLN-Mut2, and BBLN-SxxA (P-values vs. +BBLN). b , Sequence alignment of BBLN (residues 55–74) with the prototypical CAMK2 kinase domain-interacting region of GRIN2B (residues 1295–1310), and the regulatory domain residues 275–294 of CAMK2D was performed with CLUSTAL O. Potential interaction sites with CAMK2(D) kinase domain (based on ref. ) are shown below the sequence alignment. c , Interaction of BBLN with CAMK2D kinase domain residues 8–275 is strongly reduced in the presence of the GRIN2B peptide (30 μM). CAMK2D kinase domain was immobilized on anti-FLAG affinity matrix (AP) and incubated with BBLN in the absence and presence of GRIN2B peptide or control peptide. After washing steps and protein elution with SDS-PAGE sample buffer, BBLN was detected by immunoblot (IB) with anti-BBLN antibody, and CAMK2D was detected by anti-CAMK2D antibody (lower blot) (P-value vs. no peptide and +Cont. pept.). d , In vitro phosphorylation assay of BBLN, BBLN-SxxA, BBLN-Mut1 and BBLN-Mut2 by CAMK2D. Phospho-site-deficient BBLN-SxxA was not phosphorylated (BBLN wild-type set to 100 %). e , Interaction of BBLN, BBLN-SxxA, BBLN-Mut1, and BBLN-Mut2 with CAMK2D residues 8–275 was determined by binding assay. CAMK2D kinase domain was immobilized on anti-Flag affinity matrix (AP), and incubated with BBLN and different BBLN mutants. After washing steps, proteins were eluted and quantitated by immunoblot (IB) (P-value vs. BBLN, BBLN-Mut1 and BBLN-Mut2). Data are mean ± s.d ( a , d , n = 3; c , e , n = 4). P-values were determined by one-way ANOVA and Tukey’s test ( a , F(4,10) = 69.97; p = 0.03874, 0.05538, 0.000003179, 6.453e-7 vs.+BBLN; c , F(2,9) = 40.32; p = 0.00003353 and 0.0002668 + GRIN2B pept. vs. no peptide and +Cont. pept. d , F(3,8) = 64.42; p = 0.000003474, 0.00164, 0.001832 vs. BBLN; e , F(3,12) = 34.40; p = 0.000003081, 0.00002688, and 0.000255 SxxA vs. BBLN, Mut1, and Mut2).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Sequencing, Incubation, Control, SDS Page, Western Blot, In Vitro, Phosphorylation Assay, Binding Assay

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Cardiac contents of BBLN, phospho-T287-CAMK2D, and total CAMK2D were determined by immunoblot (IB) of cardiac proteins in heart specimen lysates from cyanotic and acyanotic TOF patients. The left panels show immunoblot images, and the right panels show quantitative data. The lower control immunoblot detects ATP5A1. b , Immunoblot detection of MLKL and phospho-S358-MLKL octamers in heart specimen lysates of cyanotic and acyanotic TOF patients. Data ( a , b ) are mean ± s.d. P-values were determined by the unpaired, two-tailed t-test (n = 6 patients per group, 3 females, 3 males; age cyanotic TOF patients: 23.17 ± 4.5 months; age acyanotic TOF patients: 29.5 ± 7.6 months; df=10; a , t = 6.0614, 4.4782, 0.6390; p = 0.0001218, 0.001182, 0.5372; b , t = 8.4943, 7.09; p = 0.000006939, 0.00003335). c , Correlation analyses were performed between cardiac contents of BBLN and total CAMK2D (left), BBLN and phospho-T287-CAMK2D (middle), and BBLN and phospho-S358-MLKL (right). Pearson correlation (r) and p-values (two-tailed) were determined of n = 6 cyanotic and 6 acyanotic TOF patients (left panel: p = 0.9209; middle panel: p = 0.0007209; right panel: p = 1.095e-7).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Western Blot, Control, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Upregulated cardiac inflammasome transcripts in transgenic, 8-month-old, male Tg- BBLN mice (Tg-1) compared with age- and sex-matched, nontransgenic FVB mice were identified by Gene Ontology (GO) analysis of NGS transcriptome data. The heat map shows members of the canonical inflammasome complex (GO: 0061702) and proinflammatory CAMK2D-regulated transcripts of ref. . P values were determined by MeV (unpaired, two-tailed t -test, just alpha; n = 4 mice per group; d.f. 6; P = 0.005223, 0.003382, 0.001927, 0.000451, 8.083 × 10 −7 , 0.00000128, 0.01809, 0.00009222, 0.002207, 4.136 × 10 −7 , 0.000005349 and 4.188 × 10 −7 ). b , NGS data of prototypical transcripts involved in fibrotic cardiac remodeling of 8-month-old, male Tg- BBLN mice (Tg-1) compared with age- and sex-matched nontransgenic FVB mice. P values were determined by an unpaired, two-tailed t -test ( n = 4 mice per group; d.f. 6; t = 70.86, 47.35, 40.73 and 0.7437; P = 5.316 × 10 −10 , 5.946 × 10 −9 , 1.465 × 10 −8 and 0.4851). c , Picrosirius red staining of paraffin-embedded heart sections shows prominent fibrotic cardiac remodeling of 8-month-old, male Tg- BBLN mice (Tg-1) compared with age- and sex-matched, nontransgenic FVB mice ( n = 4 mouse hearts per group; scale bar, 2 mm).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Transgenic Assay, Two Tailed Test, Staining

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Cardiac contents of DES were determined by IB analysis of cardiac lysates prepared from male, 8-month-old Tg- BBLN (Tg-2) and Tg- BBLN –SxxA mice. b , IB detection of cardiac DES was performed in male, 8-month-old Tg- BBLN mice (Tg-2) without and with lentiviral transduction of an miRNA targeting Camk2d by RNAi (+miCamk2d). IB images (left) and quantitative IB data (right) of intact DES and the major DES fragment. Control IBs detect ATP5A1. Data are mean ± s.d. ( n = 6 mice per group). P values were determined by an unpaired, two-tailed t -test (d.f. 10; a , t = 4.870 and 5.281; P = 0.0006519 and 0.0003571; b , t = 5.825 and 10.40; P = 0.0001673 and 0.000001111).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Transduction, Control, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Quantitative IB determination of cardiac contents of BBLN and activated phospho-T287–CAMK2D and total CAMK2D in male, 4-month-old B6 mice with 2 months of AAC, 4-month-old B6-miBbln mice with 2 months of AAC (AAC+miBbln) and 4-month-old sham-operated B6 mice (sham). IB images (left) and quantitative IB data (right). The lower control blot detects ATP5A1. b , IB determination of cardiac phospho-S345–MLKL (upper) and total MLKL (lower) contents of male, 4-month-old B6 mice with 2 months of AAC (AAC), 4-month-old B6-miBbln mice with 2 months of AAC (AAC+miBbln) and 4-month-old sham-operated B6 mice (sham). Predominant MLKL octamers as a feature of necroptosis were quantified. MLKL octamers (O), tetramers (T) and monomers (M) are marked with arrows. IB images (left) and quantitative IB data (right). c , Immunofluorescence colocalization of BBLN with p-S345–MLKL on heart specimens of a 4-month-old B6 mouse with 2 months of AAC (AAC), a 4-month-old B6-miBbln mouse with 2 months of AAC (AAC+miBbln) and a 4-month-old sham-operated B6 mouse (sham). The immunofluorescence is representative of four mouse hearts per group (scale bar, 40 μm). d , The left ventricular EF and the LVIDd were determined by echocardiography. Data are mean ± s.d. ( n = 6 male mice per group; one-way ANOVA and Tukey’s test; a , F (2,15) = 379.4, 354.4 and 32.97; top: P = 0, 1.246 × 10 −12 and 0.09708; middle: P = 1.727 × 10 −12 , 3.11 × 10 −13 and 0.3463; bottom: P = 0.003928, 0.000002048 and 0.001971; b , F (2,15) = 458.4 and 31.79; top: P = 0, 0 and 0.808; bottom: P = 0.00002672, 0.00000835 and 0.7711. d , F (2,15) = 28.47 and 15.68; top: P = 0.0005565, 0.000006051 and 0.05481; bottom: P = 0.0009046, 0.000402 and 0.9084.

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Control, Immunofluorescence

Journal: Molecular Vision

Article Title: Comparison of camel tear proteins between summer and winter

doi:

Figure Lengend Snippet: Comparison of 2-DE Coomassie-stained protein profiles and differential expression spots of camel tears between summer and winter. A : Tear proteins (100 μg) in the summer (Cs) and in the winter (Cw) were separated on first-dimensional pH 3–10 linear IPG gels (13 cm) and second-dimensional 13% vertical slab gels. The relative MW is given on the left, while the pI is given at the top of the figure. The spots marked by arrows and numbers were cut and digested, and then identified using MALDI-TOF/TOF-MS. B-I: Protein spots w1, w2, w3 and s7, and w7 with different volume intensities are displayed in the enlarged spot views of 2-DE images ( B - E ) and as three-dimensional images obtained by Melanie 4.0 software ( F - I ). Spots w1, w2, w3, w4, w5, w6 and s4, s5, and s6 were identified as LF and spots s7 and w7 were characterized as VMO1 homolog. B , D , F , H : The summer group (Cs); C , E , G , I : The winter group (Cw).

Article Snippet: Rabbit anti-human vitelline membrane outer layer protein 1 (VMO1) polyclonal antibody reacting against to a region within amino acids 1 to 167 of human VMO1 (GeneTex Inc., San Antonio, TX) and rabbit anti-human lactoferrin (LF) antibody reacting against amino acids 650 to the COOH-terminus of human LF (Abcam Inc., Cambridge, UK) were used for western blotting to validate the 2-DE and mass spectrum results.

Techniques: Comparison, Staining, Quantitative Proteomics, Software

Journal: Molecular Vision

Article Title: Comparison of camel tear proteins between summer and winter

doi:

Figure Lengend Snippet: Western blot analysis of decreasing expression of LF and increasing expression of VMO1 homolog in camel tears in the summer compared to the winter. A , B : Comparison of expression of LF ( A ) and VMO1 homolog ( B ) between the summer group (Cs) and the winter group (Cw) by western blotting. C , D : Relative quantitative analysis of each corresponding band of LF ( C ) and VMO1 homolog ( D ) in two groups, based on the volume intensity of the band in Cw as 1.0. The paired student’s t test was performed and showed a significant difference (*p=0.042, **p=0.002) between two groups.

Article Snippet: Rabbit anti-human vitelline membrane outer layer protein 1 (VMO1) polyclonal antibody reacting against to a region within amino acids 1 to 167 of human VMO1 (GeneTex Inc., San Antonio, TX) and rabbit anti-human lactoferrin (LF) antibody reacting against amino acids 650 to the COOH-terminus of human LF (Abcam Inc., Cambridge, UK) were used for western blotting to validate the 2-DE and mass spectrum results.

Techniques: Western Blot, Expressing, Comparison