antibodies targeting c3  (Hycult Biotech)

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status

doi: 10.1152/ajpheart.00272.2024

Figure Lengend Snippet: 2-D STORM images of cardiomyocytes analyzed for RyR2 clusters and Jph2 localizations. Representative STORM images showing a defined “region of interest” (ROI = 3 × 10 8 nm 2 ) from EU ( a ), PTU ( a ), and PTU + T3 ( a ) cardiomyocytes. Localizations are individual arbitrarily color-coded spots that represent signals in the fluorescent channels corresponding to RyR2 (green) and Jph2 (magenta) immunolabeling. RyR2 and Jph2 localizations show alignment in organized rows corresponding to sarcomere z -lines (scale bar, 1 µm). Analysis of localizations in the STORM images of a images are shown in the corresponding b images EU ( b ), PTU ( b ), and PTU + T3 ( b ) (scale bar, 1 µm). DBSCAN of RyR2 localizations are joined by green mesh, and each cluster is encircled within a 210 nm radius from the cluster centroid (blue circles). Boxed areas ( i ) in each of the b images are enlarged in EU.i, PTU.i, and PTU + T3.i (scale bar, 250 nm). RyR2 image data analyses showed that 40%–80% of total RyR2 localizations within a cell area were clustered and that ∼10% to 30% of total Jph2 localized within the defined RyR2 cluster areas. 2-D, two-dimensional; DBSCAN, density-based spatial clustering of applications with noise; EU, euthyroid; Jph2, junctophilin-2; NNDs, nearest neighbor distances; PTU, propyl-thiouracil; ROI, region of interest; RyR, ryanodine receptor; STORM, stochastic optical reconstruction microscopy; T3, triiodo- l -thyronine.

Article Snippet: For immunostaining, adherent cardiomyocytes were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed with PBS, and permeabilized and blocked with 0.2% Triton X-100 plus 5% goat serum in PBS for 1 h. Myocytes were incubated overnight at 4°C with mouse monoclonal anti-RyR2 (DyLight 550 conjugated) antibodies (1:200; NBP2-80143R/C3-33; Novus Biologicals, Centennial, CO) and rabbit polyclonal anti-Jph2 (1:400; Invitrogen 405300, Thermo Fisher Scientific) or rabbit polyclonal anti-Ca V 1.2 (1:400; ACC-003; Alomone, Jerusalem, Israel) antibodies.

Techniques: Immunolabeling, Microscopy

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status

doi: 10.1152/ajpheart.00272.2024

Figure Lengend Snippet: Histograms showing the distribution of RyR2 clusters by size. STORM images were analyzed by DBSCAN to identify RyR2 clusters and number of Jph2 colocalized with RyR2 clusters. Histogram shows RyR2 clusters segregated by size into bins of 25 localizations per cluster from 10 minimum localizations (loc) to 299 loc/cluster (displayed on x -axis). Bar graphs represent means ± SE of data averaged from 10 to 12 cells/heart of 5 to 6 animals per group [EU, PTU, and PTU + T3 (T3)]. Statistical analysis of data in each histogram used a linear mixed-effects model with Tukey’s post hoc pairwise comparisons. A : bars represent the number of RyR2 clusters ( y -axis) in each bin. B : Jph2 localizations per cluster binned by RyR2 cluster size; LME statistical analysis showed differences between PTU vs. EU ( P < 0.05) and PTU vs. T3 ( P < 0.01). C : NNDs between RyR2 clusters based on cluster size. D : relative frequencies of RyR2 clusters ( y -axis) distributed by NND between clusters ( x -axis). *NND with the highest number of RyR2 clusters from each group with majority of RyR2 clusters from EU and T3 myocytes measuring 0.3–0.4-µm NND, whereas NND of clusters in PTU cells was 0.5–0.6 µm. DBSCAN, density-based spatial clustering of applications with noise; EU, euthyroid; Jph2, junctophilin-2; LME, linear mixed-effect; NNDs, nearest neighbor distances; PTU, propyl-thiouracil; ROI, region of interest; RyR, ryanodine receptor; STORM, stochastic optical reconstruction microscopy; T3, triiodo- l -thyronine.

Article Snippet: For immunostaining, adherent cardiomyocytes were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed with PBS, and permeabilized and blocked with 0.2% Triton X-100 plus 5% goat serum in PBS for 1 h. Myocytes were incubated overnight at 4°C with mouse monoclonal anti-RyR2 (DyLight 550 conjugated) antibodies (1:200; NBP2-80143R/C3-33; Novus Biologicals, Centennial, CO) and rabbit polyclonal anti-Jph2 (1:400; Invitrogen 405300, Thermo Fisher Scientific) or rabbit polyclonal anti-Ca V 1.2 (1:400; ACC-003; Alomone, Jerusalem, Israel) antibodies.

Techniques: Microscopy

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status

doi: 10.1152/ajpheart.00272.2024

Figure Lengend Snippet: 3-D STORM images of RyR2 clusters and Jph2 localizations in ventricular myocytes. Representative 3-D STORM images of cardiomyocytes isolated from EU, PTU, and PTU + T3-treated animals. Localizations are individual arbitrarily color-coded spots that represent signals in the fluorescent channels corresponding to RyR2 (green) or Jph2 (magenta). a : subregions of an analyzed ROI (3 × 10 11 nm 3 ) are shown to illustrate a 3-D view of the image [ x , y , z planes indicated in EU ( a )] with RyR2 clusters encircled by a sphere (blue lattice) measuring 210 nm radius from the cluster centroid, and individual Jph2 are seen as spots or localizations (scale bar is 1 µm at the front of the 3-D image). b : images are higher magnification of regions in corresponding images in a images (scale bar, 600 nm) illustrating RyR2 clusters (spots joined by green mesh) and Jph2 (magenta spots) inside and outside each sphere encircling a cluster. c : images illustrate higher magnification of individual RyR2 clusters within each sphere colocalized with Jph2 (scale bar, 200 nm). 3-D, three-dimensional; EU, euthyroid; Jph2, junctophilin-2; PTU, propyl-thiouracil; ROI, region of interest; RyR, ryanodine receptor; STORM, stochastic optical reconstruction microscopy; T3, triiodo- l -thyronine.

Article Snippet: For immunostaining, adherent cardiomyocytes were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed with PBS, and permeabilized and blocked with 0.2% Triton X-100 plus 5% goat serum in PBS for 1 h. Myocytes were incubated overnight at 4°C with mouse monoclonal anti-RyR2 (DyLight 550 conjugated) antibodies (1:200; NBP2-80143R/C3-33; Novus Biologicals, Centennial, CO) and rabbit polyclonal anti-Jph2 (1:400; Invitrogen 405300, Thermo Fisher Scientific) or rabbit polyclonal anti-Ca V 1.2 (1:400; ACC-003; Alomone, Jerusalem, Israel) antibodies.

Techniques: Isolation, Microscopy

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status

doi: 10.1152/ajpheart.00272.2024

Figure Lengend Snippet: 3-D STORM images of RyR2 clusters and Ca V 1.2 in ventricular myocytes. Representative 3-D images of cardiomyocytes from the three study groups (EU, PTU, and PTU + T3) are illustrated as described in legend to showing localizations of RyR2 (green mesh) clustered within 210 nm radius spheres (blue lattice). Localizations of Ca V 1.2 (yellow spots) appear within and outside the spheres. Images in b and c are magnified subregions of the ROIs shown in corresponding a images. Scale bars are indicated. 3-D, three-dimensional; EU, euthyroid; PTU, propyl-thiouracil; ROI, region of interest; RyR, ryanodine receptor; STORM, stochastic optical reconstruction microscopy; T3, triiodo- l -thyronine.

Article Snippet: For immunostaining, adherent cardiomyocytes were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed with PBS, and permeabilized and blocked with 0.2% Triton X-100 plus 5% goat serum in PBS for 1 h. Myocytes were incubated overnight at 4°C with mouse monoclonal anti-RyR2 (DyLight 550 conjugated) antibodies (1:200; NBP2-80143R/C3-33; Novus Biologicals, Centennial, CO) and rabbit polyclonal anti-Jph2 (1:400; Invitrogen 405300, Thermo Fisher Scientific) or rabbit polyclonal anti-Ca V 1.2 (1:400; ACC-003; Alomone, Jerusalem, Israel) antibodies.

Techniques: Microscopy

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status

doi: 10.1152/ajpheart.00272.2024

Figure Lengend Snippet: Jph2 and Ca V 1.2 localizations per RyR2 cluster analyzed in 3-D STORM images. Quantitation of Jph2 and Ca V 1.2 localizations within a sphere of 210 nm radius of the RyR2 cluster centroid are shown as violin plots as described in legend to . A : Jph2 localizations within the RyR2 cluster sphere. B : percentage of total RyR2 clusters in the cell area measured (ROI volume) without any associated Jph2. C : Ca V 1.2 localizations per cluster sphere. D : percentage of all clusters without Ca V 1.2. Statistical analysis comparing study groups (EU, PTU, and PTU + T3) used one-way ANOVA, post hoc Tukey’s multiple comparisons test; P values between groups indicated by brackets. γ P = 0.06, * P < 0.05, *** P < 0.001, and **** P < 0.0001. 3-D, three-dimensional; EU, euthyroid; Jph2, junctophilin-2; PTU, propyl-thiouracil; ROI, region of interest; RyR, ryanodine receptor; STORM, stochastic optical reconstruction microscopy; T3, triiodo- l -thyronine

Article Snippet: For immunostaining, adherent cardiomyocytes were fixed in 4% paraformaldehyde (PFA) for 20 min, then washed with PBS, and permeabilized and blocked with 0.2% Triton X-100 plus 5% goat serum in PBS for 1 h. Myocytes were incubated overnight at 4°C with mouse monoclonal anti-RyR2 (DyLight 550 conjugated) antibodies (1:200; NBP2-80143R/C3-33; Novus Biologicals, Centennial, CO) and rabbit polyclonal anti-Jph2 (1:400; Invitrogen 405300, Thermo Fisher Scientific) or rabbit polyclonal anti-Ca V 1.2 (1:400; ACC-003; Alomone, Jerusalem, Israel) antibodies.

Techniques: Quantitation Assay, Microscopy

Journal: Frontiers in Immunology

Article Title: Functional and structural characterization of mouse Factor H-related B protein unveils a novel dimerization domain shared by FHR-B and FH

doi: 10.3389/fimmu.2025.1522651

Figure Lengend Snippet: FHR-B binds strongly to C3-opsonized surfaces independently of the presence of sialic acids. (A) Cryostat kidney sections from C57BL/6 and Cfh -/- mice immunostained for mC3 and FHR-B reveal that FHR-B is naturally deposited in the C3-opsonized mice glomeruli. (B) Hybrid proteins with the C-terminal region of FHR-B (FHR-1 1-3 ::FHR-B 4-5 ), FHR-C (FHR-1 1-3 ::FHR-C 14-15 ), FHR-E (FHR-1 1-3 ::FHR-E 4-5 ) and mFH (FHR-1 1-3 ::mFH 19-20 ) were added to cryostat kidney sections of Cfh -/- mice and detected with a mouse monoclonal antibody directed to the N-terminal region of FHR-1 (2C6) that is present in all these hybrid proteins. Data illustrate that the hybrid FHR-1 1-3 ::FHR-B 4-5 shows a much stronger binding to C3-opsonized surfaces than the hybrid including the C-terminal region of mFH (FHR-1 1-3 ::mFH 19-20 ). No binding was observed with FHR-1 1-3 ::FHR-C 14-15 and FHR-1 1-3 ::FHR-E 4-5 hybrids. (C) Same binding assay on cryostat kidney sections of Cfh -/- ;Cfhrs -/- mice was performed with the hybrid proteins FHR-1 1-3 ::mFH 19-20 , FHR-1 1-3 ::FHR-B 4-5 and the full length rFHR-B. Much stronger binding was again observed for FHR-1 1-3 ::FHR-B 4-5 than FHR-1 1-3 ::mFH 19-20 and this binding was similar to that obtained for the rFHR-B protein. Interestingly, binding of FHR-1 1-3 ::FHR-B 4-5 and rFHR-B, but not of FHR-1 1-3 ::mFH 19-20 was preserved after desialylation of the cryostat kidney sections using Clostridium perfringens neuraminidase. Importantly, different antibodies were used to detect the hybrid proteins (2C6 anti-FHR-1 N-terminus) and full length rFHR-B ( in-house pAb ant-FHR-B). As cryostat kidney sections of Cfh -/- ;Cfhrs -/- mice were used, the in-house pAb ant-FHR-B shows no backgorund staining.

Article Snippet: Cell opsonization was confirmed by flow cytometry experiments with an in-house monoclonal mouse anti-human C3 antibody (12.17) and a phycoerythrin-labeled goat anti-mouse IgG secondary antibody (eBiosciences, #12-4010-82).

Techniques: Binding Assay, Staining

Journal: Frontiers in Immunology

Article Title: Role of factor H-related protein 3 in Pseudomonas aeruginosa bloodstream infections

doi: 10.3389/fimmu.2024.1449003

Figure Lengend Snippet: FHR-3 increases the deposition of C3 on P. aeruginosa. FHR-3 deficient serum (25%) without (black columns) or supplemented with FHR-3 at 2 µg/ml of serum (white columns) or at 100 µg/ml of serum (grey columns) were incubated for 15 min at 37°C with PAO1 or the isogenic serum-sensitive strain PAO1Δ wzz2 . Deposition of C3 on the bacterial surface was determined by ELISA. Bars represents the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons; P values are indicated on the bars. ** P< 0.01.

Article Snippet: Next, microtiter plate wells were incubated sequentially with a mouse mAb anti-human C3 that recognizes an epitope in the ß-chain (C3-12.17) , an alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (Sigma), and developed with p-nitrophenyl phosphate (Sigma) in 50 mM carbonate-bicarbonate buffer (pH 9.6) plus 5 mM MgCl 2 .

Techniques: Incubation, Enzyme-linked Immunosorbent Assay