WT AVMs expressing FKBP-AKAR3 were treated with 100 nmol/L isoproterenol (ISO, Sigma) for 10 min as indicated. The levels of phospho-RyR2 at Ser2807 (pRyRS2807) and Ser2814 (pRyRS2814), RyR2, phospho-PKA substrate (RRXS*/T*) (pPKAsub), and FKBP-AKAR3 were detected in western blots. The treated AVMs from indicated mice were lysed with RIPA buffer supplement with proteinase and phosphatase inhibitors. Immunoblotting was applied to detect the expression of pRyR2-S2807 (ab59225, Abcam, Cambridge, MA), pRyR2-S2814 (A010-31, Badrilla, England), RyR2 (MA3-925, Thermofisher, IL), pPKAsub (9624, Cell Signaling, Danvers, MA), FKBP-AKAR3 (GFP, 632592, Clontech, CA), and γ-tubulin (T6557, Sigma-Aldrich, St Louis, MO). IRDye 680RD goat anti-rabbit IgG secondary antibody (926–68071, LI-COR, Lincoln, NE) and IRDye 800CW goat anti-mouse IgG secondary antibody (926–32210, LI-COR, Lincoln, NE) were used for multi-color detection. PVDF membranes were scanned on Biorad Chemdoc MP imaging systems (Biorad, Hercules, CA). The optical density of the bands was analyzed with NIH Image J software (https://imagej.nih.gov/ij/ ).
Ma3 925
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The MA3-925 is a laboratory instrument designed for performing general analytical measurements. It features a liquid-crystal display and keypad for user interaction. The device is intended for use in research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ma3 919, by Thermo Fisher Scientific (3 mentions)
Ab59225, by Abcam (2 mentions)
Irdye 800cw goat anti mouse igg secondary antibody, by LI COR (1 mentions)
Irdye 680rd goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
T6557, by Merck Group (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Pa1 913, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence substrate, by Merck Group (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Ab31092, by Abcam (1 mentions)
Ab3625, by Abcam (1 mentions)
Ab2864, by Abcam (1 mentions)
Ab52309, by Abcam (1 mentions)
Mab1691, by Merck Group (1 mentions)
A4700, by Merck Group (1 mentions)
A7811, by Merck Group (1 mentions)
Ab58552, by Abcam (1 mentions)
Ab2865, by Abcam (1 mentions)
A6455, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
10 protocols using ma3 925
1
RyR2 Phosphorylation Dynamics in AVMs
2
Immunofluorescence Imaging of Ryanodine Receptor and Calsequestrin in Muscle Fibers
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Intact FDB fibres were plated on extracellular matrix and cultured overnight at 37 °C. Fibres were fixed for 10 min in PBS containing 4% formalin and 100 μM EGTA and then washed 3 × 5 min in PBS. Fibres were then permeabilized for 30 min in 1% Triton X-100 and blocked for 1 h in blocking buffer (2% goat serum, 0.1% Triton X-100 and 0.5% BSA). Antibodies to RyR1 (Thermo Scientific MA3-925, 1:300 dilution) and CSQ (Thermo Scientific, PA1-913, 1:400 dilution) were diluted in blocking buffer and applied overnight at 4 °C. After washing 3 × 5 min in PBS, fibres were incubated with Alexa Fluor conjugated secondary antibodies for 2 h at room temperature before being mounted using VECTASHIELD mounting media. Fibres were imaged in the Optical Imaging and Vital Microscopy Core at BCM on a Zeiss LSM 780 confocal microscope using the × 60 oil objective and images were quantified in Image J.
3
Muscle Protein Expression Analysis
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Crude muscle homogenates were separated by electrophoresis and transferred onto membranes. Membranes were incubated with primary antibody (anti-ryanodine receptor 1 (RyR1), MA3-925, Thermo; anti-dihydropyridine receptor (DHPR) α2 subunit, ab2864, Abcam; anti-SERCA1, MA3-911, Thermo; anti-SERCA2, ab3625, Abcam; anti-actin, A4700, Sigma; anti-troponin I, MAB1691, Millipore; anti-3-NT, ab52309, Abcam; anti-malondialdehyde (MDA), MD20A-R1a, Academy Bio-Medical; anti-tumor necrosis factor α (TNF-α), 11948, Cell Signaling; anti-high-mobility group box 1 (HMGB1), 326059652, SHINO-TEST; anti-NADPH oxidase 2 catalytic subunit gp91phox (NOX2/gp91phox), ab31092, Abcam; anti-neuronal, anti-endothelial, and anti-inducible nitric oxide synthases (nNOS, eNOS, and iNOS, respectively), 610308, 610296, 610328, respectively, BD Biosciences; anti-manganese SOD (SOD2), 06-984, Upstate). Images of the membrane were collected following exposure to chemiluminescence substrate (Millipore) using a charge-coupled device camera attached to ChemiDOC MP (Bio-Rad), and Image Lab Software was used for detection as well as densitometry.
4
Comprehensive Cardiac Protein Analysis
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The following primary antibodies were used: mouse monoclonal antibodies to α-actinin [Western blotting (WB), 1:2500; immunofluorescence (IF), 1:400; A7811, Sigma-Aldrich], actin (WB, 300 ng/ml; A2171, Sigma-Aldrich), myomesin [WB/IF, 1:10; mMaC, Developmental Studies Hybridoma Bank (DSHB)], myosin (WB/IF, 1:10; MF20, DSHB), RyR2 (WB/IF, 1:1000; MA3-925, Thermo Fisher Scientific), SERCA2 (WB, 1:1000; IF, 1:100; MA3-919, Thermo Fisher Scientific), PLN [WB, 1:5000 (A010-14; Badrilla); WB, 2 μg/ml; IF, 1:200 (ab2865, Abcam)], titin (IF, 1:10; 9D10, DSHB), obscurin-NH2 (WB/IF, 1:10; tissue culture supernatant) (13 (link)), and Hax-1 (WB, 1:1000; clone 52; 610824, BD Biosciences); rabbit monoclonal antibody to HSP90 (WB, 1:1000; 4877, Cell Signaling); and rabbit polyclonal antibodies to dihydropyridine receptor (WB/IF, 1:200; ab58552, Abcam), junctophilin-2 (WB, 1 μg/ml; 40-5300, Invitrogen), titin-Z (IF, 2 μg/ml) (44 (link)), SERCA2 (IF, 1:100; Ab91032, Abcam), sAnk1.5 (WB, 300 ng/ml; IF, 3 μg/ml) (9 (link)), SERCA2-pSer38 (WB, 1:1000; IF, 100; A010-25AP, Badrilla), RyR2-pSer2808 (WB, 1:2000; IF, 1:100; ab59225, Abcam), RyR2-pSer2814 (WB, 1:1000; A010-31AP, Badrilla), PLN-pSer16 (WB, 1:5000; A010-12, Badrilla), PLN-pSer16 (IF, 1:200; 07-052, Millipore), obscurin-COOH (WB, 300 ng/ml) (9 (link)), and obscurin-Ig58/59 (IF, 3 μg/ml) (45 (link)).
5
Immunolabeling of GFP and RyR
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Paraformaldehyde-fixed cultures were double immunolabeled with the polyclonal rabbit anti-GFP antibody (serum, 1:10,000, A6455 Thermofisher Scientific) and the monoclonal mouse anti-RyR (34C, 1:500, MA3-925 Thermofisher Scientific) and with Alexa-488 and Alexa-594, respectively, as previously described (66 (link)). The 14-bit images were recorded with a cooled CCD camera (SPOT) and Metaview image processing software. Image composites were arranged in Adobe Photoshop CS6 and linear adjustments were performed to correct black level and contrast.
6
Quantitative Analysis of SR-Related Proteins
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SR‐related proteins were analyzed by Western blotting as previously described (Eshima, Tamura, et al., 2017 (link)). The protein abundance of the ryanodine receptor (RyR), the dihydropyridine (DHPR), the calsequestrin (CSQ), and the SR Ca2+‐ATPase (SERCA) was assessed. Briefly, polyvinylidene fluoride membranes were incubated overnight at 4°C with the following primary antibodies: anti‐type 1 ryanodine receptor (RyR) antibody 34C (MA3‐925; Thermo Fisher Scientific); anti‐dihydropyridine (DHPR) antibody 20A (ab2864; Abcam); anti‐calsequestrin antibody VIIID12 (MA3‐913; Thermo Scientific); anti‐SR Ca2+‐ATPase 2 (SERCA2) antibody 2A7‐A1 (MA3‐919; Thermo Scientific), and anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody 14C10 (no. 2118; Cell Signaling Technology) at 4°C. The membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase, enhanced by SuperSignal West Dura and Femto extended duration substrate (Thermo Fisher Scientific), and quantified by densitometry (C‐DiGit, LI‐COR Biosciences).
7
Immunolabeling of Cardiac Muscle Slices
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After culture, LMSs were washed in PBS and then fixed in 4% paraformaldehyde solution for 30 min at room temperature. Slices were permeabilized in 1% Triton X-100 for 1 h at room temperature. Slices were then blocked using blocking buffer (10% FBS, 5% BSA, and 10% horse serum in PBS) for 2 h at room temperature. LMSs were incubated with the primary antibody overnight at 4°C in blocking buffer and then washed three times for 30 min with PBS. LMSs were next incubated with secondary antibody in blocking buffer for 3 h at room temperature and then washed three times for 30 min with PBS. LMSs were then stored in PBS at 4°C. Imaging of immunolabeled LMS was done with a confocal microscope (Zeiss LSM780) using a 63× oil objective with numerical aperture of 1.4. Primary antibodies were as follows: mouse IgG anti–caveolin-3 (1/500; 610421; BD Biosciences), rabbit IgG anti-connexin43 (Cx43; 1/2,000; C6291; Sigma-Aldrich), mouse IgG anti-RYR (1/200; MA3-925; Thermo Fisher Scientific), and rabbit IgG anti-phosphor2814-RYR (1/200; A010-31AP; Badrilla); and secondary antibodies were as follows: goat anti-mouse IgG Alexa568 (1/2,000; Life Technologies) and goat anti-rabbit IgG Alexa488 (1/2,000; Life Technologies).
8
Immunocytochemistry of SERCA2a, RyR2, pCaMKII
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Immunocytochemistry was performed using antibodies against SERCA2a (A010-20, Badrilla), RyR2 (MA3-925, Thermo Fisher Scientific) and phospho-Thr286-CaMKII (ab32678, Abcam).9 (link) Western blotting and detailed immunostaining analyses were used to validate the specificity of antibodies. Cells pretreated with KN-93 served as a negative control for anti-phospho-Thr286-CaMKII antibody; meanwhile bona fide target staining was distinguished from the background by staining with a suitable secondary antibody alone.
9
Quantitative Analysis of Cardiac Proteins
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Atrial tissue was collected and homogenized as described previously (Alvarado et al., 2017 (link)), in a buffer containing 0.9% NaCl, 10 mM Tris-HCl pH 6.8, 20 mM NaF and protease inhibitors. Equal amounts of protein, as determined by Bradford assay, were loaded. 50 µg of tissue homogenate, in Laemmli buffer, was separated by SDS-PAGE in 4–20% TGX or AnyKD precast gels (Bio-Rad). Proteins were transferred to PVDF membrane using the iblot2 transfer system (ThermoFisher) or wet transfer. Primary antibodies were as follows: anti-RyR2 (1:2000; MA3-925, ThermoFisher), SERCA2 (1:1000; MA3-919, ThermoFisher), NCX (1:1000; MA3-926, ThermoFisher), PLN (1:5000; A010-14, Badrilla), pT17-PLN (1:5000; A010-13, Badrilla), pS16-PLN (1:5000; A010-12, Badrilla), Cav1.2 (1:200; ACC-003, Alomone), GAPDH (1:10000; MAB374, Millipore). Secondary antibodies were: goat anti-mouse-HRP (1:5000; 31437, ThermoFisher) or goat anti-rabbit-HRP (1:5000; 31463, ThermoFisher). Secondary antibody concentrations were 5x higher when using the ibind Flex system. SuperSignal ECL reagent (ThermoFisher) was used to develop membranes followed by imaging with a ChemiDoc MP apparatus (Bio-Rad). Band intensities were quantified with the ImageLab software (Bio-Rad) or using ImageJ (NIH).
10
Cardiac Protein Expression Analysis
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Protein samples were prepared from left ventricular free walls. Forty micrograms of proteins were separated on SDS-PAGE gels and transferred on nitrocellulose membranes. Membranes were blocked and incubated with primary antibodies targeted against Nav1.5 (D9J7S, Cell Signaling technology; 1:1000), SERCA2 (PA5-29380 Thermo Scientific; 1:2000), Na + /Ca 2+ exchanger NCX1 (Santa Cruz Biotechnology; 1:1000), CaMKII (PA5-22168 Thermo Scientific; 1:1000), p-CaMKII (MA1-047 Thermo Scientific; 1:2000), ox-CaMKII (GTX36254 GeneTex; 1:1000), phospholamban (PLB; Santa Cruz Biotechnology; 1:1000), pPLB-T17 (Santa Cruz Biotechnology; 1:5000), pPLB-S16 (Santa Cruz Biotechnology; 1:1000), type 2 ryanodine receptor (RyR2; MA3-925 Thermo Scientific; 1:2000), pRyR2-S2808 (A010-30 Badrilla; 1:4000), pRyR2-S2814 (A010-31 Badrilla; 1:4000), pRyR2-S2030 (A010-32 Badrilla; 1:4000), N-cadherin (4061, Cell Signaling technology; 1:1000) and calmodulin (CaM; 05-173 EMD Millipore; 1:1000). In addition, an anti-GAPDH antibody (Santa-Cruz Biotechnologies; 1:10000 dilution) was used as an external/internal control.
Next, membranes were incubated with the ad hoc secondary horseradish peroxidase (HRP) antibody (Santa Cruz; 1:10000). Incubation was followed by detection using chemiluminescence. Western-blot quantification was performed with Image Lab TM 5.2.1 software (Bio-Rad Software).
Next, membranes were incubated with the ad hoc secondary horseradish peroxidase (HRP) antibody (Santa Cruz; 1:10000). Incubation was followed by detection using chemiluminescence. Western-blot quantification was performed with Image Lab TM 5.2.1 software (Bio-Rad Software).
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