β1d-integrin antibody  (Millipore)

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 reduces surface integrin levels and destabilizes the sarcolemma. a Representative Western blots of sarcolemma protein extracts from hearts of tTA control, Thbs3 DTG, and Thbs4 DTG mice, assayed for integrin proteins and β-dystroglycan. Laminin2α2 and Cacna1c serve as loading controls. Quantitation is shown in Supplementary Figure . The red box shows integrin proteins specifically reduced on the sarcolemma by Thbs3. b Western blots for input of microsomal protein extracts before immunoprecipitation for Thbs3 and β1D integrin and Cacna1c from NRVMs infected with Adβgal or AdThbs3 (left panel). The microsomal fraction was then immunoprecipitated (IP) for β1 integrin and blotted for the indicated proteins (right panel). Mouse IgG was used as a control for the IP. c Regimen of Evans blue dye (EBD) and isoproterenol (Iso, 300 mg/kg) injection to measure membrane permeability in the hearts of mice. d Representative histological heart sections from 3 tTA control and 3 Thbs3 DTG mice, imaged for WGA (green fluorescence) and leakage of EBD into the cardiomyocytes by red fluorescence subjected to the experimental regimen shown in c . Scale bars are 1 mm. e Regimen of Evans blue dye (EBD) injection after TAC surgery to measure membrane permeability in adult mice. Mice were sacrificed (Sac) 24 h after EBD injection, 1 week after TAC surgery. f , g Representative cardiac histological images of tTA control and Thbs3 DTG hearts 1 week after TAC surgery stained with WGA (green) and intracellular leakage of EBD (red) according to the experimental scheme shown in e , while g shows quantitation of EBD positive cells in the hearts of these mice. Scale bars are 100 μm. * P < 0.05 versus tTA control sham; two-tailed students T -test. Data are represented as percentage EBD positive cells (≥3000 cells from ≥8 animals). Error bars are +/− standard error of the mean and number of mice used is shown in the graph as individual data points

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Western Blot, Control, Quantitation Assay, Immunoprecipitation, Infection, Injection, Membrane, Permeability, Fluorescence, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Loss of Thbs3 protects the heart from pressure overload pathology. a Low magnification cardiac histological images from WT and Thbs3 −/− mice stained with Masson’s trichrome 12 weeks after TAC or sham surgery. Scale bar = 1 mm. b HW/BW ratios in the indicated groups of mice 12 weeks after TAC or sham surgery. c Echocardiography measured fractional shortening (FS) percentage and d LVIDD in the indicated groups of mice 12 weeks after a TAC or sham surgical procedure. e Pulmonary edema was analyzed by LW/BW ratio measurement in the indicated groups of mice 12 weeks after a TAC or sham surgical procedure. f Percentage of fibrotic area was quantified with heart sections stained with Masson’s trichrome in WT and Thbs3 −/− mice after TAC. g Representative Western blots for integrin proteins from heart sarcolemma protein extracts from WT and Thbs3 −/− mice 12 weeks after TAC or sham surgery. Cacna1c served as loading control. Quantitation of these results is shown in Supplementary Figure . * P < 0.05 versus WT control sham; # P < 0.05 versus WT TAC. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test and two-tailed students T -test. Error bars are +/− standard error of the mean and number of mice used in each experiment are shown in the graphs

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Staining, Western Blot, Control, Quantitation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Induction of endogenous Thbs3 is detrimental after cardiac injury. a Schematic diagram depicting the Thbs gene-deleted mice used to selectively analyze effects of endogenous Thbs3. b Representative Western blots from hearts of 1–3 day-old WT, Thbs1/2/3/4/5 −/− and Thbs1/2/4/5 −/− mice for the indicated Thbs proteins. Gapdh served as loading control. c Kaplan–Meier survival plot of shams, WT, Thbs1/2/3/4/5 −/− and Thbs1/2/4/5 −/− mice after TAC surgery in days. Number of mice used is shown in the graph for each group. P < 0.0001 analyzed by log-rank test WT versus Thbs1/2/4/5 −/− and Thbs1/2/3/4/5 −/− versus Thbs1/2/4/5 −/− . d Experimental regimen of EBD and Iso injection into the groups of mice shown ( e ) to measure membrane permeability. e Quantification of EBD positive area in the hearts of the indicated groups of mice after Iso injection with the regimen shown in d . * P < 0.05 versus WT; # P < 0.05 versus Thbs1/2/4/5 −/− mice. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/− standard error of the mean and number of mice used in each experiment are shown in the graphs. f Immunohistochemistry for β1D integrin from heart sections of WT, Thbs3 −/− , Thbs1/2/4/5 −/− and Thbs1/2/3/4/5 −/− mice 1 week after TAC surgery. Scale bars are 50 μm. g Representative Western blots for the indicated integrin proteins from hearts of WT, Thbs3 −/− , Thbs1/2/4/5 −/− , and Thbs1/2/3/4/5 −/− mice subject to 1 week of TAC and processed for sarcolemma protein extracts. Cacna1c served as loading control. Quantitation of these results is shown in Supplementary Figure

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Western Blot, Control, Injection, Membrane, Permeability, Immunohistochemistry, Quantitation Assay

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Integrin overexpression reduces Thbs3-mediated membrane instability and disease. a Schematic of the breeding used to generate combinatorial Thbs3 DTG, α7β1D integrin TG mice. b Representative Western blots for Thbs3 and the indicated integrin proteins from heart sarcolemma protein extracts from tTA, Thbs3 DTG, α7β1D integrin TG and Thbs3 DTG/ α7β1D integrin TG mice. The α1Na + /K + -ATPase served as loading control. c Quantification of EBD positive area from cardiac histological sections after Iso (300 mg/kg) injection in Thbs3 DTG, α7β1D integrin TG, Thbs4 DTG, and Thbs3 DTG/ α7β1D integrin TG mice. d Representative histological images of heart sections from tTA, Thbs3 DTG and Thbs3 DTG/ α7β1D integrin TG mice stained with WGA-FITC (green) after Iso injection (300 mg/kg). EBD is shown as red fluorescence. Scale bars are 300 μm. e Fractional shortening (FS) percentage as determined by echocardiography following 2 weeks of continuous Iso infusion (60 mg/kg/day) or PBS vehicle controls. Number of mice analyzed is shown within each histogram in c , e . * P < 0.05 versus vehicle treated; #<0.05 versus Thbs3 DTG with Iso. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/− standard error of the mean

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Over Expression, Membrane, Western Blot, Control, Injection, Staining, Fluorescence

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 enhances secretory pathway activity but reduces membrane integrins. a Time course of intracellular trafficking fluorescence changes in cultured neonatal ventricular cardiomyocytes (NRVMs) infected with a GalNac-T2-RFP baculovirus and the indicated adenoviruses. The data show a quantitative time course of GalNac-T2-RFP recovery in the Golgi network after FRAP to measure ER-to-Golgi vesicular trafficking. * P < 0.05 versus Adβgal infected cells. b Quantitative time course of loss of VSVG-eGFP fluorescence in the Golgi after iFRAP as a measurement for Golgi-to-membrane (Golgi exit) vesicular trafficking. * P < 0.05 versus Adβgal infected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. c Western blots for ECM proteins using heart extracellular matrix extracts from tTA, Thbs3 DTG and Thbs4 DTG mice at baseline or with TAC stimulation. A silver-stained gel is shown with a non-specific (n.s.) band as a loading control. d , e Quantitative time-course of the loss of α5 integrin-GFP fluorescence at the Golgi after iFRAP as a measure of Golgi-to-plasma membrane vesicular trafficking of α5 integrin. The data were generated by iFRAP of α5 integrin-GFP from COS-7 cells d transfected with the plasmids harboring the cDNAs shown or e treated with recombinant Thbs3 or Thbs4 proteins, or bovine serum albumin (BSA) as a control. * P < 0.05 versus Adβgal transfected cells; #<0.05 versus Thbs3 transfected cells. Statistical analysis was performed using two-way ANOVA and Turkey multiple comparisons test. Results were summed from four independent experiments and error bars are +/− standard error of the mean

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Activity Assay, Membrane, Fluorescence, Cell Culture, Infection, Western Blot, Staining, Control, Clinical Proteomics, Generated, Transfection, Recombinant

Journal: Nature Communications

Article Title: Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability

doi: 10.1038/s41467-018-08026-8

Figure Lengend Snippet: Thbs3 enhances integrin turnover. a – h Cell surface biotinylated proteins were used for input control experiments or immunoprecipitated with streptavidin (Surface, a – d ) and endocytic protein uptake was induced for 30 min followed by immunoprecipitation with streptavidin (Uptake, e – h ). Quantitative analysis of protein band intensities of β1D integrin, α5 integrin, calreticulin (CRT), and calnexin (CXN) from NRVMs infected with Adβgal, AdThbs3, AdThbs4, AdThbs3-RGD adenovirus or treated with recombinant Thbs3 protein (western blots used for quantification are presented in the uncropped western blot images attachment). Data are represented as fold expression over Adβgal relative to input controls. * P < 0.05 versus Adβgal. Statistical analysis was performed using one-way ANOVA and Turkey multiple comparisons test. Error bars are +/− standard error of the mean and number of independent experiments are shown in the graphs

Article Snippet: Primary antibody incubations were performed overnight at 4 °C: integrin β1D (EMD Millipore, #MAB1900 at 1:100), PDI (Abcam, #ab2792 at 1:100), Thbs3 (Proteintech, #19727-1-AP at 1:100).

Techniques: Control, Immunoprecipitation, Infection, Recombinant, Western Blot, Expressing