Hearts from the euthanized mice were fixed in 4% formaldehyde (pH 7.3) for 12 h, dehydrated in alcohol, clarified in xylene, and embedded in paraffin to be sectioned at 5 μm. Protein levels were evaluated in cardiac tissues using the immunoperoxidase technique with anti-p-MYPT1 (T696) (Cell Signaling, USA #5163), anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029) antibodies. Staining was performed using a peroxidase and diaminobenzidine kit with a chromophore according to the manufacturer’s instructions (RTU-Vectastain kit; Vector Laboratories, USA). The heart tissue was additionally stained with hematoxylin. The images were obtained with a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan) and analyzed with ImageJ software (ImageJ 1.47v).
Anti rock2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-ROCK2 is a monoclonal antibody that specifically recognizes the ROCK2 protein. ROCK2 is a serine/threonine protein kinase that plays a key role in the regulation of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Anti rock1, by Cell Signaling Technology (5 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti p mypt1 t696, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Rtu vectastain kit, by Vector Laboratories (1 mentions)
Eclipse 400 epifluorescence microscope, by Nikon (1 mentions)
Hrp conjugated goat anti rabbit, by Cell Signaling Technology (1 mentions)
Anti aldh1a2, by Abcam (1 mentions)
Anti cd45, by BD (1 mentions)
Anti ho 1, by Cell Signaling Technology (1 mentions)
Anti crmp2, by Cell Signaling Technology (1 mentions)
Cy3 conjugated anti rabbit secondary antibody, by Abcam (1 mentions)
Y 27632, by Merck Group (1 mentions)
Anti mypt1, by Cell Signaling Technology (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p p65 ser536, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti rhoa, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
10 protocols using anti rock2
1
Evaluating Cardiac Protein Levels
2
Comprehensive Western Blot Analysis Protocol
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The western blot protocol was conducted as previously described [18 (link)]. The antibodies used in the study were as follows: anti-CD45 (BD Biosciences, 610266, (1/1000), anti-ROCK2 (Cell Signaling, 1/1000), SLC45A3 (Abcam, ab137065, 1/1000), and anti-ALDH1A2 (Abcam, ab156019, 1/2000). Unfortunately, there is no available specific antibodies for KCNK3 [19 (link), 20 (link)], thus avoiding any measurement of the protein expression. When tested in kcnk3-knockout miceand Kcnk3-mutated rats, we previously found that all commercially anti-KCNK3 antibodies tested were unusable [20 (link)]. This is why we analyzed KCNK3 expression by RT-qPCR. The blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse diluted 1:10,000 (Cell Signaling) or with HRP-conjugated goat anti-rabbit diluted 1:5000 (Cell Signaling). The antibodies were detected using ECL reagents. (Perkin–Elmer). ImageJ software was used to quantify the level of protein expression.
3
Western Blot Analysis of Neuronal Proteins
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Brain tissues and N2a cells were lysed using low-temperature RIPA buffer (CWBIO, Beijing). 10 μg of total protein was resolved on 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene (PVDF) membranes. Membranes were probed with the primary antibodies including anti-Rock2 (9029; Cell Signaling Technology), anti-endothelial nitric oxide synthase (eNOS; 32027; Cell Signaling Technology), anti-HO-1 (43966; Cell Signaling Technology), anti-Nrf2 (ab89443; Abcam), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab181602, Abcam) antibodies. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibodies, and protein signals were observed with ECL reagents.
4
Nogo-66 Protein Purification and Antibody Detection
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Soluble Nogo-66 protein was obtained via SUMO fusion in Escherichia coli from our laboratory (16 (link)). Y-27632 and the NEP1-40 were obtained from Merck KGaA and Tocris Bioscience, respectively. The Cy3-conjugated anti-rabbit secondary antibody was obtained from Abcam (cat. no. ab6939). Rabbit monoclonal IgG anti-APP was purchased from LifeSpan BioSciences, Inc. In addition, the following primary antibodies were obtained: Anti-β-secretase 1 (BACE1; cat. no. 5606; Cell Signaling Technology, Inc.), anti-ROCK2 (cat. no. ab125025; Abcam), anti-phosphorylated (p)-collapsin response mediator protein-2 (CRMP2; Thr514; cat. no. 9397; Cell Signaling Technology, Inc.), anti-CRMP2 (cat. no. 35672; Cell Signaling Technology, Inc.), anti-GAPDH (cat. no. BS72410; Bioworld Technology, Inc.) and anti-microtubule-associated protein 2 (MAP2; cat. no. 05-346; Merck KGaA). HRP-conjugated goat anti-rabbit IgG antibody (cat. no. E030120-01; Earthox Life Sciences) was used. All reagents and drugs used were of analytical grade.
5
Western Blot Analysis of ROCK and NF-kB Signaling
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U937 cells were washed with PBS and lysed as described previously (27 (link)). Total protein concentration was quantified with Bradford Assay. Extracted proteins were separated by SDS-PAGE (100 V), and transferred to a nitrocellulose membrane (110 V, 90 min). Membranes were blocked in 5% (w/v) non-fat milk powder in TBS-T for 1 h at room temperature (RT), washed with TBS-T, and incubated with primary antibody overnight at 4°C. Membranes were washed and incubated with HRP-conjugated secondary antibodies for 2 h at RT, then washed and incubated with enhanced chemiluminescence (ECL) reagents (Sigma-Aldrich, USA) according to the manufacturer’s instructions. The primary antibodies used were anti-ROCK1 (Cell Signaling, USA #4035), anti-ROCK2 (Cell Signaling, USA #9029), anti-MYPT1 (Cell Signaling, USA #2634), anti-pMYPT1 (T696) (Cell Signaling, USA #5163), anti-p65 (Cell Signaling, USA #8242), anti-p-p65(Ser536) (Cell Signaling, USA #3033), anti-GAPDH (Cell Signaling, USA #2118), and anti-β-actin (Cell Signaling, USA #8457). Secondary antibodies used were anti-mouse-HRP (Santa Cruz Biotechnology, USA SC-516102) and anti-rabbit-HRP (Santa Cruz Biotechnology, USA SC-2357).
6
Western Blot of Cell Signaling Proteins
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Western blot analyses were performed according to a standard protocol using primary antibodies, including anti-ZXDC (Proteintech, 20503-1AP, 1:800), anti-RhoA(Cell Signaling Technology, #2117, 1:1000), anti-ROCK1 (Cell Signaling Technology, #4035, 1:1000), anti-ROCK2 (Cell Signaling Technology, #8236, 1:1000), anti-IGF2BP3 (Proteintech, 14642-1-AP, 1:2000), anti-p-CofilinS3 (Cell Signaling Technology, #3313, 1:1000), antiCofilin (Cell Signaling Technology, #5175, 1:2000), anti-p-MLC2S19 (Cell Signaling Technology, #3671,1:500), and anti-MLC2 (Cell Signaling Technology, # 8505, 1:1000) antibodies. GAPDH (Cell Signaling Technology, #5174) and α-Tubulin (Sigma-Aldrich, T9026) were used as loading controls.
7
Protein Quantification and Western Blotting
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Total proteins were collected using RIPA buffer (Thermo Scientific, CA, USA) and then quantified for protein quantification by using Pierce™ BCA Protein Quantification Kit (Thermo Scientific) [15 (link)]. Protein (20 μg) was used for electrophoresis loading SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibodies, including anti-KIAA1429 (Cell Signaling Technology, 1:1000, #88,358), anti-ROCK2 (Cell Signaling Technology, 1:1000, #47,012), and beta-actin (CWBio, Beijing, China). After incubation by primary antibodies or their corresponding secondary antibodies, blots were developed using SuperSignal West Dura Persistence Substrate (Thermo Scientific).
8
Western Blot Analysis of EMT Regulators
9
Vimentin-mediated Oxidative Stress Signaling
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Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit and oxLDL were from BiYuntian Biological Technology Institution (Shanghai, China). Y27632 (inhibits ROCK1 and ROCK2 with equal potency) was obtained from Selleckchem (Houston, TX, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and trypsin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-vimentin, anti-ROCK1, anti-ROCK2, anti-caspase3, anti-GAPDH, anti-caspase8, anti-caspase9, anti-p-vimentin (Ser56), anti-p-vimentin (Ser83), anti-ICAM-1, and anti-VCAM-1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-and Cy3-conjugated anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-vimentin and fluorescein (FITC)-conjugated anti-mouse IgG antibodies were purchased from Boster Company (Wuhan, China).
10
Western Blot Antibodies and Materials
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Ether was purchased from Lek (Ljubljana, Slovenia). Luminol and p-coumaric acid were obtained from Sigma Aldrich Corporation (St. Louis, MO, USA). Protease (Complete, Ultra Mini, EDTA-free) and phosphatase inhibitor cocktails (PhosStop), were purchased from Roche (Mannheim, Germany). The rabbit polyclonal antibodies (anti-Rho A, anti-phospho-α1
Na + /K + -ATPase (Ser 23 ) and anti-α1Na + /K + -ATPase) and monoclonal (anti-PI3K p85α) were obtained from Abcam (Cambridge, UK). The rabbit polyclonal (anti-ROCK2) and monoclonal (anti-PI3K p110α) antibodies were purchased from Cell Signalling Technology (CST, USA). The goat polyclonal anti-α2 Na + /K + -ATPase antibody, rabbit polyclonal mouse anti-actin monoclonal antibody, and the secondary anti-mouse and anti-rabbit antibodies conjugated to alkaline phosphatase (ALP) or to horseradish peroxidase (HRP) and BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium chloride), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Na + /K + -ATPase (Ser 23 ) and anti-α1Na + /K + -ATPase) and monoclonal (anti-PI3K p85α) were obtained from Abcam (Cambridge, UK). The rabbit polyclonal (anti-ROCK2) and monoclonal (anti-PI3K p110α) antibodies were purchased from Cell Signalling Technology (CST, USA). The goat polyclonal anti-α2 Na + /K + -ATPase antibody, rabbit polyclonal mouse anti-actin monoclonal antibody, and the secondary anti-mouse and anti-rabbit antibodies conjugated to alkaline phosphatase (ALP) or to horseradish peroxidase (HRP) and BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium chloride), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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