Heart samples were obtained after reperfusion for 2 h and were immediately stored at -80°C before western blot analyses were performed. Protein was extracted using the RIPA protein lysate (ASPEN, Wuhan, China), and the concentration of the protein was determined using the BCA protein concentration assay kit (ASPEN, Wuhan, China). Subsequently, protein (40 μg) was separated by 10% SDS-PAGE and electrotransferred to PVDF membranes. After incubation with corresponding antibodies, the PVDF membranes were then washed by TBST and incubated with a secondary antibody for 1 h at room temperature. A chemiluminescence method was used to detect protein bands, and AlphaEaseFC software processing system (Alpha Innotech, USA) was used to analyze the optical density of target bands. The following primary antibodies were used for western blot analysis: Bax (1 : 2000), p-ASK1 (1 : 500), ASK1 (1 : 1000), NF-κB p65 (1 : 1000), histone H3 (1 : 3000), p-JNK (1 : 1000), and JNK (1 : 2000) (Cell Signaling Technology, USA); Bcl-2 (1 : 2000), caspase 3 (1 : 2000), p-CaMKII (1 : 1000), CaMKII (1 : 500), TNF-α (1 : 1000), IL-6 (1 : 500), IL-1β (1 : 500), and RyR2 (1 : 500), GAPDH (1 : 10000) (Abcam, UK); ox-CaMKII (1 : 500) (GeneTex, USA); p-RyR2 (Ser2814, 1 : 500) (Badrilla, UK); and cleaved caspase 3 (1 : 1000) (Affbiotech, USA).
P camkii
Manufactured by Abcam
Sourced in United States, United Kingdom
P-CaMKII is a phosphorylated form of the calcium/calmodulin-dependent protein kinase II (CaMKII) enzyme. CaMKII plays a crucial role in various cellular processes, including synaptic plasticity, memory formation, and calcium signaling. The phosphorylated form, P-CaMKII, is a marker for the active state of the enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Camkii, by Abcam (6 mentions)
Gapdh, by Abcam (3 mentions)
P creb, by Abcam (3 mentions)
Caspase 3, by Abcam (2 mentions)
P acc, by Cell Signaling Technology (2 mentions)
P ask1, by Cell Signaling Technology (1 mentions)
Bca protein concentration assay kit, by Aspen (1 mentions)
Ox camkii, by GeneTex (1 mentions)
Tnf α, by Abcam (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Il 1β, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Nf κb, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
13 protocols using p camkii
1
Cardiac Protein Expression Analysis
2
Protein Expression Analysis of Dorsal Root Ganglia
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The cryopreserved tissues of the dorsal root ganglia were lysed using a protease lysis buffer and centrifuged to obtain supernatants. Following protein concentration measurement using the BCA method, the proteins were subjected to SDS-PAGE, transferred to a membrane, sealed in skim milk for 2 h and incubated with antibodies against nuclear factor-kappa B (NF-κB) (Abcam, USA, 1:1000), CaMKII (Abcam, USA, 1:1000), p-CaMKII (Abcam, USA, 1:1000), CREB (Abcam, USA, 1:1000), p-CREB (Abcam, USA, 1:1000) and GAPDH (Abcam, USA, 1:1000) at 4°C overnight. This step was followed by 3 times membrane washing and incubation with an HRP-labeled goat anti-rabbit IgG antibody at 37°C for 1 h. Finally, the color of the proteins was developed using an ECL kit and a gel imaging system. The absorbance was analyzed by the ImageJ software.13 (link)
3
Western Blot Analysis of Signaling Proteins
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The cultured PAECs were lysed with ice-cold lysis buffer (Tris 50Mm, pH 7.4, NaCl 150 mM, Triton X-100 1%, glycerol 10%, Nonidet P-40 1%, EDTA 5 mM, and PMSF 2mM) and centrifuged at 13,500 r.p.m for 15 min at 4°C. The supernatant was collected and total protein was determined using BCA protein assay kit with bovine serum albumin as the standard (Beyotime Institute of Biotechnology, China). Samples with equal amount of protein (50 μg) were separated in 10%-12% SDS-PAGE and then transferred onto nitrocellulose membranes (Millipore, MA). The membranes were then blocked with 5% fat-free milk in Tris-buffered saline and 1% Tween-20 (TBS-T) for 1 h and then incubated with the corresponding antibodies overnight at 4°C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody, for 2h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) kit (Pierce, CA) and exposed on an X-ray film. The immunoblots intensities were quantified using Quantity One software (BioRad). All antibodies were purchased from Santa Cruz except Kir1.1, Kir2.2, p-CaMKII and CaMKII from Abcam.
4
Multimodal Protein Profiling in Cells
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After the stimulation period, cells were fixed in 4% PFA, stained for specific antigens using primary antibodies against desmin (DAKO), DDR2 (Santa Cruz), NCX (SWANT), P4HB, Vimentin, P‐CaMKII, ADAMTS13 (Abcam), incubated with fluorophore‐conjugated secondary antibodies and visualized on Zeiss LSM 510 Meta and Nikon A1 confocal microscopes. The images were analyzed using ImageJ software (ImageJ 1.46r, NIH, USA).
5
Biflorin Neuroprotective Effects Protocol
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Biflorin was provided by one of the authors (D.S. Jang) and suspended in 10% Tween 80 solution. Donepezil hydrochloride monohydrate, scopolamine hydrobromide, and dizocilpine (MK-801) were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C-ζ (PKC-ζ), phosphorylated PKC-ζ at Thr 410, cAMP response element-binding protein (CREB) at Ser 133, extracellular signal-regulated kinase (ERK), and phosphorylated ERK at Thr202/Tyr204 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against phosphorylated CaMKII at Thr 286 (pCaMKII) was purchased from Abcam (Cambridge, UK). All other materials were obtained from normal commercial sources and were of the highest grade available. All drugs were freshly made on the day of testing. Donepezil, a positive control, and scopolamine were dissolved in a 0.9% saline solution.
6
Western Blot Analysis of Signaling Proteins
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Cultured cells and whole tissues extracts were prepared with RIPA buffer supplemented with protease inhibitor cocktail (MERCK P8340), and phosphatase inhibitor cocktails (MERCK P5726 and P0044). Western blotting was performed using the Mini-PROTEAN Tetra Cell electrophoresis system, and transferred to PVDF membranes. The following primary antibodies concentrations were used p-CaMKII 1:1000 (Abcam ab182647), CaMKII 1:1000 (Cell Signaling 3362), GAPDH 1:10,000 (Abcam ab181602), p-ERK1/2 1:20,000 (MERCK M9692), ERK1/2 1:40,000 (MERCK M5670), p-RXR 1:1000 (Affinity Biosciences), RXR antibody 1:200 (SCBT sc-553), p-RYR 1:2000(Abcam ab59225), RYR 1:1000 (ab2868), p-Rac1 1:1000 (Millipore 07-896-I) Rac1 1:1000 (Millipore 05-389), (and Vinculin (provided by Benny Geiger, Weizmann Institute of Science). Horseradish peroxidase conjugated secondary anti-mouse, anti-rabbit or anti-goat was used to detect proteins (Jackson Immunology). Western blots were imaged using the Chemidoc Multiplex system (Bio-rad) and Image Lab software (Bio-rad).
7
Protein Expression Analysis in RAW264.7 Cells and Chondrocytes
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Total proteins of RAW264.7 cells and primary chondrocytes were acquired by the RIPA lysis buffer (#R0010, Solarbio, China) with the supplement of 1 mM phosphatase inhibitor cocktail (#B15002, Bimake, USA) and 1 mM phenylmethanesulfonyl fluoride (#329-98-6, Solarbio). Western blot was executed as previously described [9 (link)]. The primary antibodies used in this work were: p-CaMKII (#ab124880, Abcam, USA), CaMKII (#ab134041, Abcam), iNos (#13120S, Cell Signaling Technology, USA), Cox2 (#12282S, Cell Signaling Technology), Col II (#BA0533, Boster, Wuhan, China), Mmp13 (#GB11247, Servicebio, China), Fth1 (#4393, Cell Signaling Technology), Gpx4 (#ab125066, Abcam), Cd206 (#18704-1-AP, Proteintech, China), Arg-1 (#16001-1-AP, Proteintech), and Gapdh (#5174, Cell Signaling Technology). After the incubation of horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibodies (#BL003A or #BL001A, Biosharp), western blots were imaged by the ChemiDocXRS + Imaging System (Tanon, Shanghai, China). Quantification of proteins was performed by the ImageJ software (version 1.8.0). All primary and secondary antibodies used in this work were commercial.
8
Protein Expression Analysis Protocol
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Cells were lysed with RIPA buffer (65 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) with protease inhibitor (04693132001, Roche) and phosphatase inhibitor tablets (4906837001, Roche). Protein concentration was quantified with a BCA kit. Similar amounts of protein were separated by 8-12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore), and incubated overnight at 4 °C with corresponding primary antibodies: F-actin (1:1000, Abcam, #ab130935), G-actin (1:1000, Abcam, #ab200046), p-CaMKII (1:1000, Abcam, #ab124880), cofilin (1:1000, Abcam, #ab54532), CaMKII (1:1000, Abcam, #ab52476), p-cofilin (1:1000, Abcam, #ab283500), ET-1 (1:1000, Abcam, #ab178454), eNOS (1:1000, Abcam, #ab199956), p-eNOS (1:1000, Abcam, #ab215717), SGLT2 (:1000, Abcam, #ab37296), Fak (1:1000, Abcam, #ab40794), Src (1:1000, Abcam, #ab133283), GAPDH (1:1000, Abcam, #ab8245), XO (1:1000, Abcam, #ab109235), SERCA2 (1:1000, Abcam, #ab150435), Fak (1:1000, Abcam, #ab40794), Src (1:1000, Abcam, #ab133283), Tom20 (1:1000, Abcam, #ab186735), Drp1 (1:1000, Abcam, #ab184247), and SERCA2 C674-SO3H (#A300-BL2103; Bethyl Laboratories, Inc., Montgomery, TX, USA). Suitable secondary antibodies were next applied for 1 h at room temperature. Signals were detected on a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).
9
Immunohistochemical Analysis of Mouse Thyroid Tumors
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Tumors sections were processed and stained as described previously (30 (link)) with the following antibodies: p-ACC (#3661-Cell signaling), p-P70S6K1 (#ab129230 Abcam), p-LKB1 (S-428) (#ab138386-Abcam), LKB1 (#ab185734 Abcam), STRAD-α (#ab192789 Abcam) p-CAMKII (#ab32678 Abcam), HADHA (#PA5-27348 Thermo Scientific), HSD17β4 (#OAGA1102-Aviva systems biology). For mouse analyses, thyroid sections from 3-5 independent mouse tumors from each genotype were analyzed, with consistent findings observed within each group.
10
Hippocampal Molecular Analyses in Behavior
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