Journal: Brain Research

Article Title: Knockout of the ?-aminobutyric acid receptor subunit ?4 reduces functional ?-containing extrasynaptic receptors in hippocampal pyramidal cells at the onset of puberty

doi: 10.1016/j.brainres.2012.02.035

Figure Lengend Snippet: Figure 4

Article Snippet: The α4 antibody utilized in our experiments was purchased from Santa Cruz Biotechnology (catalog #sc-7355).

Techniques:

Journal: Scientific Reports

Article Title: Exosomes from Adipose-Derived Stem Cells (ADSCs) Overexpressing miR-21 Promote Vascularization of Endothelial Cells

doi: 10.1038/s41598-019-49339-y

Figure Lengend Snippet: Characterization of ADSCs isolated from inguinal fat pad of 3-week old Lewis male rats by flow cytometric analysis. ( A – D ) Flow cytometric analysis of ADSC surface markers (CD29, CD44, CD49d and CD34). The results presented are typical of those obtained from three separate experiments. ( E ) Summary of the result in ( A – D ). Error bars were calculated based on triplicates. ( *** means P < 0.001 vs. isotypic control).

Article Snippet: Akt was obtained from CST (#4685), CD49D from Miltenyi (#130-111-487), CD63, Calnexin, CD81 from SANTA (SC-15363, SC-70481, SC7637).

Techniques: Isolation, Control

Journal: International Journal of Biological Sciences

Article Title: Inhibition of α4β1 Integrin Activity by Small Tellurium Compounds Regulates PD-L1 Expression and Enhances Antitumor Effects

doi: 10.7150/ijbs.95350

Figure Lengend Snippet: Role of VLA-4 integrin in PD-L1 expression downregulation by SAS. ( A ) B16/F10 melanoma cells or ( B ) D122 cells were cultured on vascular cell adhesion molecule-1 (VCAM-1)- or bovine serum albumin (BSA)-coated plates with or without SAS for 1 h. The cells were washed twice . The percentage of attached cells, representing VLA-4 (very late antigen- 4 ) activity, was determined by the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) viability test relative to the control PBS. *p<0.001 vs. BSA; #p<0.01 vs. PBS; **p<0.001 vs. PBS. Significance was calculated via one-way ANOVA. The results are presented as the mean+SE of 3 experiments. ( C ) Human VLA-4-negative AML cells isolated from AML patients were cultured on FN-coated plates for 24 hours. The cells were collected and stained with PE-conjugated anti-mouse PD-L1. ( D ) B16/F10 melanoma cells were transfected with either control or (E) VLA-4 shRNA (short hairpin RNA) and cultured with or without different concentrations of SAS. The cells were collected and stained with either PE-conjugated anti-mouse PD-L1 antibodies or isotype-matched controls. The results show one representative of 3 experiments. Figs C, D and E are accompanied by quantitative data from flow cytometry analysis. # p<0.001 vs. the isotype control; *p<0.05 vs. the PBS group. **p<0.01 vs. PBS.

Article Snippet: USA); paclitaxel (Sigma Aldrich); p65-GFP-RelA (Addgene; Watertown, MA, USA); p239-AKT (NIH); VLA-4 shRNA, mouse integrin α4 shRNA, and control plasmid (Santa Cruz Biotechnology; Texas, USA).

Techniques: Expressing, Cell Culture, Activity Assay, Control, Isolation, Staining, Transfection, shRNA, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Inhibition of α4β1 Integrin Activity by Small Tellurium Compounds Regulates PD-L1 Expression and Enhances Antitumor Effects

doi: 10.7150/ijbs.95350

Figure Lengend Snippet: SAS inhibits both PD-L1 expression and VLA-4 activity in U937 myelomonocytic human leukemic cells. ( A ) U937 human cells were cultured in the presence or absence of SAS. The cells were collected and stained with either PE-conjugated anti-human PD-L1 antibodies or their respective isotype-matched controls. The results show one representative experiment of 3 performed. ( B ) U937 cells were cultured on VCAM-1- or BSA-coated plates with or without SAS for 1 h. The cells were subsequently washed twice. The percentage of attached cells (representing VLA-4 activity) was determined by the XTT viability test relative to the control PBS. * p<0.001 vs. BSA **p<0.001 vs. PBS. Significance was calculated via one-way ANOVA. The results represent the mean +SE from 3 experiments ( B ). ( C ) The VLA-4 conformational structure was detected by FRET. VLA-4 activation involves the separation of the fluorescently tagged cytoplasmic ends of the α- and β-subunits of the integrin, resulting in a reduction in the FRET signal. U937 cells were transfected with α4-mCFP and β1-mYFP. After 48 hours, the cells were activated and fixed. FRET of the molecular interaction between the cytoplasmic domains of α4 and β1 was determined as described in the Materials and Methods. ( D ) Results for FRET positive controls (cells expressing CFP and YFP encoded on the same plasmid (i.e., maximal FRET obtained in this system) and negative controls (CFP and YFP expressed by different plasmids and undergoing minimal FRET, i.e., only that produced by random colocalizations). *p<0.01 vs. the negative control. Significance was calculated via a two-tailed t test. PLL (poly-L-lysine). FN (Fibronectin).

Article Snippet: USA); paclitaxel (Sigma Aldrich); p65-GFP-RelA (Addgene; Watertown, MA, USA); p239-AKT (NIH); VLA-4 shRNA, mouse integrin α4 shRNA, and control plasmid (Santa Cruz Biotechnology; Texas, USA).

Techniques: Expressing, Activity Assay, Cell Culture, Staining, Control, Activation Assay, Transfection, Plasmid Preparation, Produced, Negative Control, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Inhibition of α4β1 Integrin Activity by Small Tellurium Compounds Regulates PD-L1 Expression and Enhances Antitumor Effects

doi: 10.7150/ijbs.95350

Figure Lengend Snippet: Associations between VLA-4, IL-10 and PD-L1. ( A ) B16/F10 cells were cultured on FN-coated plates in the presence or absence of SAS at various concentrations. The cells were collected, fixed, permeabilized and stained with PE-conjugated αIL-10 Abs. ( B ) SK-MEL23 cells were cultured on FN-coated plates in the presence or absence of SAS at various concentrations, with or without IFNγ (2 μg/ml), for 48 h. The supernatants were collected, and IL-10 levels were measured by ELISA. *p<0.0001 vs. without IFNγ; **p<0.001 vs. PBS. Significance was calculated via two-way ANOVA. The results represent the mean+SE of 3 experiments. SK-MEL23 cells without ( C, E ) or with IFNγ ( D, F ) were cultured on FN-coated plates in the presence or absence of SAS at various concentrations for 24 h, after which the cells were collected and stained for PD-L1 ( C, D ) or VLA-4 ( E, F ). All FACS results show one representative of 3 experiments. ( G ) SK-MEL23 cells were cultured in the presence or absence of SAS at various concentrations with or without IFNγ (2 μg/ml) for 24 h. The cells were detached and replated again on VCAM-1- or BSA-coated plates with or without SAS at various concentrations for 1 h. The cells were subsequently washed twice . The percentage of attached cells (representing VLA-4 activity) was determined by the XTT viability test relative to the control. *p<0.0001 vs. without IFNγ; **p<0.001 vs. PBS. Significance was calculated via two-way ANOVA. The results represent the mean+SE from 3 experiments. VLA-4 (Very late antigen-4/integrin α4β1). IFNγ (interferon gamma).

Article Snippet: USA); paclitaxel (Sigma Aldrich); p65-GFP-RelA (Addgene; Watertown, MA, USA); p239-AKT (NIH); VLA-4 shRNA, mouse integrin α4 shRNA, and control plasmid (Santa Cruz Biotechnology; Texas, USA).

Techniques: Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Fatty-Acid-Binding Protein 4 as a Novel Contributor to Mononuclear Cell Activation and Endothelial Cell Dysfunction in Atherosclerosis

doi: 10.3390/ijms21239245

Figure Lengend Snippet: FABP4 inhibition suppressed the oxidized-LDL-induced adhesiveness of MNCs by modulating the adhesion molecules. Oxidized-LDL enhanced the FABP4 expression in MNCs from control subjects ( A ). The oxidized-LDL (50 µg/mL)-induced adhesiveness of MNCs was reversed by FABP4 neutralizing antibody ( B ). The expression of adhesion molecules, such as intergrin β2, integrin α4, and P-selectin glycoprotein ligand 1 (PSGL-1), was higher in MNCs from CAD patients ( C ). The oxidized-LDL-induced adhesiveness of MNCs was reversed by integrin β2, integrin α4, and PSGL-1 neutralizing antibodies ( D ). FABP4 neutralizing antibody reduced the oxidized-LDL-induced integrin β2, integrin α4, and PSGL-1 expression in MNCs ( E ). N represents normal subjects; Pt represents CAD patients; B represents baseline. N = 6 in each experiment. Data are presented as means ± standard errors. Statistical analyses were performed using Student’s t test. Data were considered statistically significant when the p -value was <0.05. * p < 0.05, # p < 0.01.

Article Snippet: Membranes were probed with monoclonal antibody directed to β-actin, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), signal transducer and activator of transcription 1 (STAT-1), phosphorylation of endothelial nitric oxide synthase (p-eNOS), stromal cell-derived factor 1 (SDF-1), FABP4 (Cell Signaling, Beverly, MA, USA), P-selectin, intergrin β2, integrin α4, and P-selectin glycoprotein ligand 1 (PSGL-1) (Santa Cruz, Dallas, TX, USA).

Techniques: Inhibition, Expressing, Control

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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

Techniques: Western Blot, Quantitation Assay, Immunoprecipitation, Infection, Injection, 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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

Techniques: Staining, Western Blot, 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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

Techniques: Western Blot, Injection, 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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

Techniques: Over Expression, Western Blot, 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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

Techniques: Activity Assay, Fluorescence, Cell Culture, Infection, Western Blot, Staining, 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 antibodies used were: glyceraldehyde 3-phosphate dehydrogenase (Gapdh (Fitzgerald, #10R-G109A at 1:10000)), Armet (Abcam, #ab67271 at 1:1000), ATF6α (Abcam, #ab37149 at 1:1000), calreticulin (Cell Signaling Technology, #2891; at 1:1000), calnexin (Abcam, #ab75801 at 1:1000), GRP78/BiP (Sigma-Aldrich, #G8918 at 1:1000), PDI (Cell Signaling Technology, #2446; at 1:1000), integrin β1D (EMD Millipore, #MAB1900 at 1:1000), integrin β3 (Cell Signaling Technology, #4702; at 1:1000), integrin α2 (Lifespan Biosciences, #LS-C159934 at 1:1000), integrin α4 (EMD Millipore, #AB1924 at 1:1000), integrin α5 (EMD Millipore, #AB1928 at 1:1000), integrin α6 (Cell Signaling Technology, #3750 at 1:1000), integrin α7 (Santa Cruz Biotechnology, #sc-27706 at 1:100), integrin α9 (Abcam, #ab140599 at 1:1000), integrin α10 (EMD Millipore, #AB6030 at 1:1000), Thbs1 (Lifespan Biosciences, #LS-B4155 at 1:1000), Thbs2 (BD Bioscience, #611150 at 1:1000), Thbs3 (Proteintech, #19727-1-AP at 1:1000), Thbs4 (Santa Cruz Biotechnology, #sc-7657 at 1:500), COMP/Thbs5 (Proteintech, #13641-1-AP at 1:1000), laminin2α2 (Sigma, #L0063 at 1:1000), β-tubulin (Licor, #926-42211 at 1:1000), Cacna1 (Alomone Labs, #ACC-022 at 1:1000), sodium-potassium ATPase (Abcam, #ab76020 at 1:1000).

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