primary antibodies anti-serca2a  (Badrilla Inc)

Journal: JCI insight

Article Title: Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

doi: 10.1172/jci.insight.166713

Figure Lengend Snippet: Figure 2. Exogenous cBIN1 normalizes membrane microdomains at t-tubules. (A) Representative transmission electron microscopy images of post- treatment hearts (scale bar: 1 μm) from each group (left). Quantification of the degree of contour of t-tubules (n = 100–101 t-tubules from 16–40 images of 2–3 myocardial sections and 3 hearts from each group). Data are presented as percentage of t-tubules. χ2 test was used to compare t-tubule contour between groups. *** indicates P < 0.001 for db/m + GFP versus db/db + GFP; ††† indicates P < 0.001 for db/db + GFP versus db/db + cBIN1. (B) Western blots of cBIN1, SERCA2a, CaV1.2, RyR2, GLUT4, and IRAP in total cardiac microsome and sucrose-gradient isolated TT/jSR fraction (F4) from each group (n = 5 hearts per group). (C) Representative spinning disc confocal images of posttreatment mouse myocardium with power spectrum analysis of boxed areas, and quantification of SERCA2a peak power density at t-tubules (n = 35–44 cells from 3 hearts per group). Nonparametric Kruskal-Wallis test fol- lowed by Dunn’s test was used for comparison between selected pairs. *, **, *** indicates P < 0.05, 0.01, and 0.001, respectively, for comparison versus db/m + GFP; †, ††, ††† indicates P < 0.05, 0.01, 0.001, respectively, for comparison between db/db + GFP and db/db + cBIN1.

Article Snippet: For V5, SERCA2a, GLUT4, and IRAP labeling, permeabilized and blocked tissue sections or fixed cardiomyocytes were incubated with primary antibodies against V5 (V8317-2MC, Sigma-Aldrich), SERCA2a (ab2861, Abcam), GLUT4 (4670-1725GA, Bio-Rad Laboratories), or IRAP (6918S, Cell Signaling Technology) overnight at 4°C.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Western Blot, Isolation, Comparison

Journal: JCI insight

Article Title: Cardiac gene therapy treats diabetic cardiomyopathy and lowers blood glucose.

doi: 10.1172/jci.insight.166713

Figure Lengend Snippet: Figure 5. AAV9-cBIN1 normalizes cardiac proteomics in diabetic mice. (A) Bar graphs of LFQ LC-MS/MS data (fold changes over control db/m mice) of SERCA2a, RyR2, GLUT4, and IRAP from the db/m mice treated with AAV9-GFP (n = 4 hearts) and db/db mice treated with AAV9-GFP (n = 4 hearts) or cBIN1 (n = 3 hearts). (B) PCA plot of all 3 groups (n = 2 repeats/heart × 3–4 hearts/group) generated based on LFQ LC-MS/MS proteomics. All data are presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s test or Kruskal-Wallis test followed by Dunn’s test was used for comparison between the selected pairs. * indicates P < 0.05 for comparison versus db/m + GFP; † indicates P < 0.05 for comparison between db/db + GFP and db/db + cBIN1.

Article Snippet: For V5, SERCA2a, GLUT4, and IRAP labeling, permeabilized and blocked tissue sections or fixed cardiomyocytes were incubated with primary antibodies against V5 (V8317-2MC, Sigma-Aldrich), SERCA2a (ab2861, Abcam), GLUT4 (4670-1725GA, Bio-Rad Laboratories), or IRAP (6918S, Cell Signaling Technology) overnight at 4°C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Control, Generated, Comparison

Journal: Physiological reports

Article Title: Muscle-specific sirtuin 3 overexpression does not attenuate the pathological effects of high-fat/high-sucrose feeding but does enhance cardiac SERCA2a activity.

doi: 10.14814/phy2.14961

Figure Lengend Snippet: FIGURE 5 Representative Western blots and graphs depicting LV protein level of SERCA2a and acetylated-SERCA2a from WT or SIRT3TG mice following 4-months of control- or HFHS-feeding. (a) SERCA2a. (b) Acetylated-SERCA2a following immunoprecipitation of acetylated-lysine containing proteins and detection of SERCA2a by Western blotting. # indicates a main effect of diet (p ≤ 0.05). Compared using a two-way ANOVA and a Tukey post-hoc test (SERCA2a: n = 10 mice per group; Acetylated-SERCA2a: n = 6 mice per group). Graphs are presented as the mean ± SE

Article Snippet: Bound acetylated- lysine containing proteins were eluted using bead elution buffer (Cytoskeleton) and acetylated- SERCA2a was detected on polyvinylidene difluoride (PVDF) membranes (Bio- Rad Laboratories) by western blotting with an anti- SERCA2a primary antibody (catalogue no. 4388, Cell Signaling Technology; 1:1000).

Techniques: Western Blot, Control, Immunoprecipitation

Journal: Physiological reports

Article Title: Muscle-specific sirtuin 3 overexpression does not attenuate the pathological effects of high-fat/high-sucrose feeding but does enhance cardiac SERCA2a activity.

doi: 10.14814/phy2.14961

Figure Lengend Snippet: FIGURE 8 SERCA2a activity and the Ca2+-dependent kinetic properties of SERCA2a in the LV of WT or SIRT3TG mice following 4-months of control- or HFHS-feeding. (a) SERCA2a activity-pCa curves displaying LV SERCA2a ATP hydrolysis over Ca2+ concentrations ranging from a pCa of 7.66 to 5.76 for WT or SIRT3TG mice following 4-months of control-feeding. (b) SERCA2a activity-pCa curves displaying LV SERCA2a ATP hydrolysis over Ca2+ concentrations ranging from a pCa of 7.66 to 5.76 for WT or SIRT3TG mice following 4-months of HFHS-feeding. (c) SERCA2a Vmax (maximal enzyme activity). (d) SERCA2a Ca50 ([Ca2+] eliciting 50% of Vmax). (e) SERCA2a Hill coefficient (slope of the relationship between [Ca2+] and enzyme activity for 10%–90% of Vmax). * indicates a main effect of genotype (p ≤ 0.05). † indicates an interaction between genotype and diet (p ≤ 0.05). Compared using a two-way ANOVA and a Tukey post-hoc test (n = 12 mice per group). Graphs are presented as the mean ± SE

Article Snippet: Bound acetylated- lysine containing proteins were eluted using bead elution buffer (Cytoskeleton) and acetylated- SERCA2a was detected on polyvinylidene difluoride (PVDF) membranes (Bio- Rad Laboratories) by western blotting with an anti- SERCA2a primary antibody (catalogue no. 4388, Cell Signaling Technology; 1:1000).

Techniques: Activity Assay, Control

Journal: Cardiovascular research

Article Title: Electrical remodelling precedes heart failure in an endothelin-1-induced model of cardiomyopathy.

doi: 10.1093/cvr/cvq351

Figure Lengend Snippet: Figure 4 Reduced LV Cx43, Cx40, and Nav1.5 expression in mice as early as 4 weeks after ET-1 induction. mRNA and protein expression of SERCA2a (A and B), Cav1.2 (C and D), Cx45 (E and F), Cx40 (G and H ), Nav1.5 (I and J), and Cx43 (K and L); as well as hET-1 mRNA (M), p-Cx43/total Cx43 protein (N), IL-6 mRNA (O), and representative western blots of LV of BT and NBT mice within Groups I–III (P). n ¼ 6–8 for qRT–PCR, and n ¼ 3–6 for western blot, for each genotype per group. *P , 0.05, **P , 0.01. SERCA2a, sarcoplasmic reticulum Ca2+– ATPase; Cav1.2, a1C–subunit of the L-type Ca2+ channel; Cx, connexin; Nav1.5, a-subunit of the cardiac Na+ channel; hET-1, human–ET-1; IL-6, interleukin-6.

Article Snippet: Twenty micrograms of protein extract were run on 4–12% SDS–PAGE, transferred to PVDF membrane, blocked in 5% non-fat dry milk in TBS-T (Tris base, NaCl, 0.1% Tween 20, pH 7.6) for 1 h, and incubated overnight at 48C in TBS-T with 5% BSA containing primary rabbit Abs against Cx43 and phospho-Cx43 (p-Cx43) (Ser368; Cell Signaling, Danvers, MA, USA), Cx45 (Invitrogen), ovem ber 13, 2015 D ow nloaded from Cx40 (Millipore, Billerica, MA, USA), Nav1.5 (Sigma-Aldrich), and Cav1.2 (Alomone Laboratories, Jerusalem, Israel), or a primary mouse Ab against SERCA2a (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR