The protein was separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, USA), and blocked with 5% non-fat dry milk in TBST. After washing three times with TBST, the following primary antibodies dissolved in antibody buffer (Keygentec, China) were used: anti-RNase L (Abcam, USA), anti-phosphor-H2A.X (Serl39) (Millipore, USA), anti-phosphor-H2B (Serl4) (Millipore, USA), anti-H2A.X (Abcam, USA), anti-H2B (Abcam, USA), anti-ROCK-1 (Abcam, USA), anti-Caspase-3 (Cell Signaling Technology, USA), ECLhCZIBPBHhf-iFeN8r/" target="_blank">anti-PARP (Cell Signaling Technology, USA), anti-p21 (Cell Signaling Technology, USA), and anti-β-actin (Cell Signaling Technology, USA). After the secondary antibody incubation, the membrane was washed three times with TBST and exposed with ECL (Millipore, USA). The corresponding semi-quantitative analysis was performed by measuring the optical density using ImageJ software.
Anti rock1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-ROCK1 is a lab equipment product that targets the ROCK1 protein. ROCK1 is a serine/threonine-protein kinase that plays a role in the regulation of the actin cytoskeleton. The product is designed for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti rhoa, by Abcam (5 mentions)
Anti collagen 1, by Abcam (4 mentions)
Anti rock2, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti f actin, by Abcam (2 mentions)
Anti nox4, by Abcam (2 mentions)
Anti timp1, by Abcam (2 mentions)
Anti mmp1, by Abcam (2 mentions)
Anti α sma, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti actin, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti h2ax, by Abcam (1 mentions)
Anti h2b, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti p21, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Antibody buffer, by Keygen Biotech (1 mentions)
18 protocols using anti rock1
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis for Protein Expression
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All the cells were harvested and lysed with ice-cold RIPA buffer (Merck Millipore, Burlington, MA, USA), protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail (Sigma-Aldrich). Equal amounts (30 μg) of protein were resolved through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). After blocking with 5% skim milk for 1 h, the PVDF membranes were incubated with primary antibodies, such as anti-VEGF 165A (Abcam PLC, Cambridge, England, UK), anti-ROCK1 (Abcam PLC, Cambridge, England, UK), anti-Met (Cell Signaling Technology, Danvers, MA, USA), anti-HIF-1α (Cell Signaling Technology), anti-Mcl-1 (Cell Signaling Technology), and anti-β-Actin (Sigma-Aldrich), overnight at 4° C. Following washing with tris-buffered saline containing Tween 20 (TBS-Tween 20), the PVDF membranes were incubated with the secondary antibody at room temperature for 1 h. After the membranes were washed, immunoreactive proteins were detected using a SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
3
Paclitaxel-Induced Vascular Permeability
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The following reagents were obtained from the indicated suppliers: Paclitaxel injection (5 mL: 30 mg, the excipients in the paclitaxel injection are polyoxyl 35 castor oil, anhydrous citric acid and anhydrous ethanol, Beijing Union Pharmaceutical Factory, Beijing, CHN), relevant vehicle (polyoxyl 35 castor oil and anhydrous ethanol, 1:1 v/v, Beijing Union Pharmaceutical Factory, Beijing, CHN), compound 48/80 (Sigma-Aldrich, Louis, MO, USA), Evans blue (Sinopharm Chemical Reagent Co., Ltd., Shanghai, CHN), and Cobra venom factor (Shanxi Powerdone Pharmaceutics Co., Shanxi, CHN).
The following antibodies were used in this study: anti-p-MYPT1 (Thr 696) (rabbit polyclonal, 5163, Cell Signaling Technology (CST), MA, USA); anti-MYPT1 (rabbit polyclonal, 2634, CST, MA, USA), anti-p-MLC2 (Thr18/Ser19) (rabbit polyclonal, 3674, CST, MA, USA), anti-MLC2 (rabbit polyclonal, 3672, CST, MA, USA), Anti-RhoA (67B9) (rabbit monoclonal, 2117, CST, MA, USA), anti-ROCK1 (rabbit monoclonal, EP786Y, Abcam, Cambridge, UK), anti-GAPDH (rabbit polyclonal, FL-335, Santa Cruz Biotechnology, CA, USA) and the secondary goat anti-rabbit IgG (H + L) antibody (ZSGB-BIO, Beijing, CHN).
The following antibodies were used in this study: anti-p-MYPT1 (Thr 696) (rabbit polyclonal, 5163, Cell Signaling Technology (CST), MA, USA); anti-MYPT1 (rabbit polyclonal, 2634, CST, MA, USA), anti-p-MLC2 (Thr18/Ser19) (rabbit polyclonal, 3674, CST, MA, USA), anti-MLC2 (rabbit polyclonal, 3672, CST, MA, USA), Anti-RhoA (67B9) (rabbit monoclonal, 2117, CST, MA, USA), anti-ROCK1 (rabbit monoclonal, EP786Y, Abcam, Cambridge, UK), anti-GAPDH (rabbit polyclonal, FL-335, Santa Cruz Biotechnology, CA, USA) and the secondary goat anti-rabbit IgG (H + L) antibody (ZSGB-BIO, Beijing, CHN).
4
Synthetic Elastin Peptides for Cell Research
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Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor® 568, CellTrace Calcein Red-Orange, AM and DiOC183 were from Invitrogen.
5
Western Blot Analysis of Apoptotic Signaling
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Proteins were extracted from cell lysates using RIPA lysis Buffer, and the protein concentration was measured using the Enhanced BCA Protein Assay Kit (Beyotime) following to the manufacturer's recommendations. Proteins were denatured by heat and then separated by SDS‐PAGE electrophoresis, finally transferred to PVDF membranes. The membranes were blocked using 1% bovine serum albumin solution for 1 hour at room temperature and then incubated with primary antibodies, including anti‐cleaved caspase‐3 (1:1000; Abcam), anti‐ROCK1 (1:1000; Abcam), anti‐phospho‐PTEN (1:1000; Abcam), anti‐PTEN (1:1000; Abcam), anti‐phospho‐AKT (1:1000; Abcam), anti‐AKT (1:1000; Abcam), anti‐phospho‐GSK‐3β (1:1000; Abcam), anti‐GSK‐3β (1:1000; Abcam) and anti‐β‐actin (1:1000; Zhongshan Jinqiao Biotechnology) at 4°C overnight, followed by incubation with HRP‐conjugated secondary antibody (1:5000; Zhongshan Jinqiao Biotechnology) at room temperature for 30 minutes. Protein bands were detected using a Immun‐Star HRPKit (Bio‐Rad) following to the manufacturer's protocols. Relative densitometry was analysed using Image J2x analysis software (NIH).
6
Protein Expression Analysis of Ischemic Cortex
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The tissues corresponding to the peri-ischemic cortex were taken out and then homogenized in the RIPA lysis buffer containing 1 mM PMSF. After centrifuging at 12,000 rpm for 10 min at 4°C, a bicinchoninic acid (BCA) protein assay kit (Bi yuntian Biotech. Co., Ltd., China) was used to determine the protein concentration of the supernatant. The supernatant was diluted by loading buffer to 1 μg/μL and then heated at 100°C for 5 min. Proteins (20 μg/well) were separated by SDA-PAGE and transferred to a PVDF membrane. The membranes were blocked for 2 h at room temperature in TBST containing 5% BSA, and then, it was incubated with specific primary antibodies overnight at 4°C: anti-bax, anti-bcl-2, anti-caspase 3, anti-ROCK1, anti-MLC, anti-p-MLC, anti-F-actin and anti-NMMHC IIA (diluted at 1:1000, Abcam, UK). After the membrane was washed with TBST, it was incubated for 2 h with a secondary antibody (goat anti-rabbit 1:10,000 Biomorld Technology, USA). Images were detected with ECL and imaged using the Gel Imaging System (BioRad, Hercules, CA, USA). Each experiment was performed with three independent replicates.
7
Western Blot Analysis of Apoptosis Regulators
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Total proteins were extracted from cells or tissues using RIPA lysis buffer (Sigma) on ice. The concentration was determined by a BCA detecting kit (Beyotime Biotechnology, Nanjing, China) according to the manufacturer’s instructions. Protein samples were separated by 10% SDS-PAGE, transferred onto a PVDF membrane (Millipore, Billerica, USA) and blocked with 5% skimmed milk powder solution for 2 h at room temperature. Next, membranes were incubated with primary antibodies: anti-caspase-3 antibody (Abcam, Cambridge, UK; dilution rates of 1:500), anti-ROCK1 (Abcam, dilution rates of 1:2000), and anti-GAPDH antibody (Abcam, dilution rates of 1:2000) at 4 °C overnight, respectively. The target proteins were then incubated with secondary HRP anti-Rabbit antibodies (Abcam, dilution rates of 1:2000) for 1 h at room temperature and visualized using an enhanced chemiluminescence detection system (Pierce, Rockford, USA). The signals were captured and intensities of bands were quantified using Image Lab™ Software (Bio-Rad).
8
STAT3 Inhibitors and Fibrosis Regulation
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WXWH0265 was provided by WuXi AppTec Group (Shanghai, China) and prepared in vivo in 5% dimethyl sulphoxide (DMSO) and 95% double‐distilled water. The DMSO concentration was limited to 0.1% in vitro. The signal transducer and activator of transcription 3 (STAT3) inhibitors, napabucasin (BBI608), fasudil (HY‐10341A), pirfenidone (HY‐B0673) and GSK429286A (HY‐11000), were purchased from MedChemExpress. S7936, Belumosudil (KD025) was purchased from Selleck Chemicals, USA. Clodronate liposomes were obtained from Liposomes (CP‐005‐005). Anti‐STAT3 (# 12640) and anti‐p‐STAT3 (Y705; # 9145) antibodies were purchased from Cell Signaling Technology (Cambridge, MA, USA). Anti‐α‐SMA (# MAB1420) antibodies were obtained from R&D Systems (Minneapolis, MN, USA). Anti‐ROCK1 (Cat# ab134181), anti‐ROCK2 (Cat# ab71598) and anti‐collagen‐Ⅰ (Cat# ab138492) antibodies were purchased from Abcam (Cambridge, UK). Anti‐β‐tubulin antibodies were purchased from Huabio Technology (Beijing, China).
9
Western Blot Analysis of Signaling Proteins
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The procedure was conducted as previously described.14 Briefly, proteins were extracted with radioimmunoprecipitation assay lysis buffer (Sigma) and electrophoretically transferred onto PVDF membranes (Amresco). The membranes were incubated first with special primary antibodies that, respectively, probed ROCK1 (anti‐ROCK1, Abcam, dilution rates of 1:2000), lysophosphatidic acid acyltransferase β (anti‐LPAATβ, Abcam, concentration of 1 µg/mL), and tyrosine kinase non‐receptor 2 (anti‐TNK2, Abcam, dilution rates of 1:50) at 4°C overnight and then with secondary antibodies (Abcam, dilution rates of 1:2000) at 25°C for 1 hour on the following day. Protein bands were detected on an X‐ray film using an enhanced chemiluminescence detection system.
10
Transfection of mimic-miR-130a in Endothelial Cells
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Transfection of mimic-miR-130a (10 nM) was performed on ECs using HiPerfect Transfection method (Qiagen, Valencia, USA)63 (link). Total RNA from untreated ECs, ECs transfected with mimic-miR-130a, treated with sEV with high (D17, D18, D20) or low miR-130a content (D1, D2, D4) was extracted using All-in-One Purification kit (Norgen, Thorold, ON Canada). miR-Scramble (Scr) was used as transfection reference sample. The expression of miR-130a was evaluated by RT-PCR63 (link). miR-130a targets were analyzed by Western Blot analysis. Proteins were obtained using RIPA buffer, as previously described63 (link). Anti-KDR (1:200, Santa Cruz), anti-ROCK1 (1:200, Abcam), and anti-HOXA5 (1:200, Abcam) antibody, together with anti-βactin, were used (1:200, Santa Cruz).
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