The HUVECs on Flat, HP1, HP2, and HP3 GHS for 2 days were washed twice with PBS and disrupted with 1 × cell lysis buffer (9803, Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma). A quantitative analysis of the samples was performed using the Bradford assay dye reagent (500–0006, Bio-Rad). The sample protein (10 μg) was boiled in 1 × loading dye for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel with sodium dodecyl sulfate. After transfer to a polyvinylidene fluoride membrane (ISEQ. 00010, Millipore), the membranes were blocked with 5% bovine serum albumin containing 1 × TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3 M) at RT for 1 h. The membranes were incubated with anti-ROCK1 (1:1000; ab45171, Abcam), anti ROCK2 (1:1000; ab71598, Abcam), and anti-GAPDH (1:2000; G8795, Sigma) antibodies at RT for 2 h. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz) in TBST at RT for 1 h. Chemiluminescence was visualized with the ECL Plus reagent (32132, Thermo Fisher Scientific) and recorded on X-ray film.
Ab71598
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab71598 is a primary antibody product offered by Abcam. It is a recombinant monoclonal antibody that targets a specific protein or antigen. The core function of this product is to facilitate the detection and analysis of the target molecule in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab45171, by Abcam (5 mentions)
Ab54835, by Abcam (3 mentions)
Ab187027, by Abcam (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Ab8227, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Ab14106, by Abcam (2 mentions)
Ab53219, by Abcam (2 mentions)
Ab46794, by Abcam (2 mentions)
Ab52642, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
P7626, by Merck Group (1 mentions)
Ecl plus reagent, by Thermo Fisher Scientific (1 mentions)
Bradford assay dye reagent, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Iseq00010, by Merck Group (1 mentions)
G8795, by Merck Group (1 mentions)
Rock2, by Abcam (1 mentions)
20 protocols using ab71598
1
ROCK1/2 Expression in HUVECs under GHS Conditions
2
Western Blot Analysis of Rho Signaling
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Lung adenocarcinoma cells were harvested and extracted using radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China). The extracted cells were boiled in loading buffer, and then 12% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to separate the equal amount of cell extracts. Then, the isolated protein samples were transferred onto the polyvinylidene fluoride membranes. According to the instructions, the membranes were cultivated with the primary antibodies at 4°C overnight, followed by secondary antibodies at room temperature for 2 h. An enhanced chemiluminescence Western blot detection kit was adopted to visualize the immunoreactive bands based on the kit instructions. The GAPDH antibody was utilized for normalization. The primary antibodies are exhibited as follows: RhoA (Abcam, Cambridge, UK, ab54835); rho associated coiled-coil containing protein kinase 1 (ROCK1, Abcam, ab45171); rho associated coiled-coil containing protein kinase 2 (ROCK2, Abcam, ab71598); GAPDH (Abcam, ab8245).
3
Protein Expression Analysis of HUVECs
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Total proteins were extracted from the HUVECs and quantified using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal proteins (20 µg per lane) were then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and loaded onto polyvinylidene fluoride (PVDF) membranes (Beyotime Institute of Biotechnology). The membranes were subjected to blocking buffer (5% skim milk) at room temperature for 1 h and probed with the primary antibodies, including anti-interleukin (IL)-6 (ab6672; 1/1,000), anti-IL-1β (ab226918; 1/1,000), anti-β-actin (ab8227; 1/2,000) and anti-ROCK2 (ab71598; 1/1,000) (all from Abcam) at 4°C overnight. The membranes were then probed with the secondary antibodies (ab205718; 1/5,000; Abcam) at room temperature for 2 h. Finally, the enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was adopted to visualize the protein blots, and the blot bands were then quantified using ImageJ software (version 1.46; National Institutes of Health).
4
Western Blot Analysis of Cellular Proteins
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Total protein was extracted using RIPA lysis buffer (Beyotime, Guangzhou, China), and the protein concentration was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated by SDS‐PAGE, then transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were subsequently incubated with primary antibodies against HNRNPC (1: 1000, Santa Cruz Biotechnology, sc‐32308, Dallas, TX, USA), RhoA (1: 3000, Abcam, ab54835, Cambridge, UK), ROCK2 (1: 1000, Abcam, ab71598), p‐γ‐H2AX (1: 1000, S139; Abcam, ab26350), α‐SMA (1: 500, Abcam, ab7817), FAP (1: 500, Santa Cruz Biotechnology, sc‐65398) and YAP (1: 1000, Abcam, ab56701), followed by incubation with secondary antibodies bound to horseradish peroxidase. Immunoreactivity was visualized using an ECL Western Blot system (Millipore, Burlington, MA, USA). β‐actin was used as an internal loading control.
5
Immunohistochemical Analysis of ROCK2
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Tumor tissues were first fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μm-thick sections were cut. Sections were blocked with 10% goat serum and incubated with an anti-ROCK2 (1:1000, #ab71598, Abcam) antibody at 4 °C overnight. Finally, images were acquired for analysis.
6
Protein Extraction and Western Blotting
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Total protein from tissue samples and RAW264.7 cells were obtained by
using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA,
USA). Samples were fractionated using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and were transferred onto a
polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA).
Membranes were blocked for 1 hour and were then incubated with
ghrelin-, ROCK2-, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific antibodies at 4°C overnight as follows: anti-ghrelin
antibody (1: 250; ab129383; Abcam, Cambridge, MA, USA), anti-ROCK2
antibody (1 µg/mL; ab71598; Abcam), and anti-GAPDH antibody (1/10000;
a181602; Abcam). The next day, membranes were incubated with secondary
antibodies (Abcam; dilution of 1: 2000) at 25°C for 1 hour. Protein
bands were detected on X-ray film using an enhanced chemiluminescence
detection system (ECL Western Blotting Substrate Kit; Abcam).
using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA,
USA). Samples were fractionated using sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and were transferred onto a
polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA).
Membranes were blocked for 1 hour and were then incubated with
ghrelin-, ROCK2-, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific antibodies at 4°C overnight as follows: anti-ghrelin
antibody (1: 250; ab129383; Abcam, Cambridge, MA, USA), anti-ROCK2
antibody (1 µg/mL; ab71598; Abcam), and anti-GAPDH antibody (1/10000;
a181602; Abcam). The next day, membranes were incubated with secondary
antibodies (Abcam; dilution of 1: 2000) at 25°C for 1 hour. Protein
bands were detected on X-ray film using an enhanced chemiluminescence
detection system (ECL Western Blotting Substrate Kit; Abcam).
7
Quantifying ROCK1 and ROCK2 in Lymph Nodes
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Formalin-fixed, paraffin wax-embedded blocks from each case were selected for immunohistochemical studies using the antibodies against ROCK1 and ROCK2. Hematoxylin and eosin-stained lymph node tissue slides were used for immunohistochemistry. Control tissue sections were made from the lymph node biopsies of the healthy subjects. Sections of 4 µm were cut from paraffin-embedded tissue blocks onto silane-coated slides. Sections were heated to 60 °C for 20 min prior to deparaffinization with xylene solution. Sections were then stained using the Bond Polymer Refine Detection Kit (Bond #DS9800) in an automated slide processing system (Bond-Max, Leica Microsystems, Buffalo Grove, IL, USA). ROCK1 (rabbit monoclonal, EP786Y, ab45171, Abcam, Cambridge, UK) and ROCK2 (rabbit polyclonal, ab71598, Abcam, Cambridge, UK) were used for ROCK1 and ROCK2 immunostaining, respectively. The percentage of cells staining was evaluated and intensity (–, +, ++, or +++) was scored from 0 to 3 [20 (link)].
8
Western Blot Analysis of Autophagy Markers
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Radioimmunoprecipitation assay (RIPA) buffer was used for protein extraction. The supernatants from cell lysates were run on 10% acrylamide gels by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore). Antibodies against ROCK2 (1:1000, #ab71598, Abcam), LC3 (1:1000, #12741, Cell Signaling Technology), p62 (1:1000, #ab56416, Abcam), and Beclin1 (1:1000, #ab210498, Abcam) and an HRP-conjugated secondary antibody (1:2000) were employed for western blotting. A chemiluminescence western blotting detection system (Bio-Rad) was used for protein detection.
9
Quantifying Apoptotic Proteins in HUVECs
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Total protein in HUVECs was extracted by using RIPA buffer (Thermo Fisher Scientific). After protein quantification, equal amount of protein was subjected to SDS-PAGE gel electrophoresis, transferred to PVDF membrane (Millipore, Bedford, MA, USA), and incubated with primary antibody against Bcl-2 (ab185002, Abcam, Cambridge, UK), Bax (ab32503, Abcam), ROCK1 (ab45171, Abcam), ROCK2 (ab71598, Abcam), or GAPDH (ab181602, Abcam). After that, the membrane was further incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (ab6721, Abcam). Specific protein signals were tested using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) and quantified using Image J software.
10
Protein Immunoblotting from Cultured Cells
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