Antibodies plasmids and chemicals used in the study were listed: GAPDH (sc-47724, Santa cruz biotechnology, for western blotting), YAP1 (ab205270, abcam, for western blotting; sc-376830, Santa cruz biotechnology, for Immunofluorescence (IF) and Immunohistochemistry), p-YAP(Ser127) (ab76252, abcam, for western blotting), CYR61 (26689-1-AP, proteintch, for western blotting), c-Myc (ab32072, abcam, for western blotting), CyclinD1 (ab16663, abcam, for western blotting), LaminB1 (PB9611, Boster, for western blotting), RhoA (#2117, CST, for western blotting), ROCK1 (ab134181, abcam, for western blotting), PKA C-ɑ(#4782, CST, for western blotting), TGR5 (ab72608, abcam, for western blotting, for IF and Immunohistochemistry), and Ki67 (#9449, CST, for Immunohistochemistry). Ursodeoxycholic acid was obtained from Target Mol (Shanghai, China), while INT-777 was obtained from Medchemexpress. TGR5 receptor antogonist SBI-115, H89, and SQ22536 were purchased from Selleck, while KT5720 was purchased from Sigma.
Ab134181
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab134181 is a laboratory reagent. It is a high-quality antibody designed for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab187027, by Abcam (4 mentions)
Ab16663, by Abcam (3 mentions)
Kt5720, by Merck Group (2 mentions)
Ab76252, by Abcam (2 mentions)
Ab72608, by Abcam (2 mentions)
Ab205270, by Abcam (2 mentions)
Int 777, by MedChemExpress (2 mentions)
Sc 376830, by Santa Cruz Biotechnology (2 mentions)
Pb9611, by Boster Bio (2 mentions)
Ursodeoxycholic acid, by Targetmol (2 mentions)
Sbi 115, by Selleck Chemicals (2 mentions)
Sq22536, by Selleck Chemicals (2 mentions)
Sc 47724, by Santa Cruz Biotechnology (2 mentions)
Ab32072, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab125025, by Abcam (2 mentions)
Ab97726, by Abcam (1 mentions)
Ab191393, by Abcam (1 mentions)
Ab124964, by Abcam (1 mentions)
Ab138492, by Abcam (1 mentions)
16 protocols using ab134181
1
TGR5 Signaling Pathway Regulation
2
Immunohistochemistry of ROCK1 and NF-κB in Rat Hippocampus
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Briefly, paraffin-embedded rat hippocampal tissue sections (thickness of 5 μm) were deparaffined, hydrated, and treated with 3% H2O2 for 10 min to block endogenous peroxidase activity. Non-specific binding was offset by 5% bovine serum albumin (BSA). Next, the sections were incubated with primary antibodies to ROCK1 (1:100, ab134181, Abcam, Cambridge, UK) or phosphorylated NF-κB p65 (phospho-S529) (1:100, ab97726, Abcam) for 2 h at room temperature, and with secondary goat anti-rabbit IgG H&L (horseradish peroxide, 1:2000, ab205718, Abcam) for 30 min, followed by another a 30-min incubation with streptavidin-horseradish peroxidase complex. The sections were then stained by diaminobenzidine, counterstained with hematoxylin, fixed, and observed under a microscope with 4 visual fields randomly selected. The positive rate was measured by ImageJ.
3
Western Blot Analysis of Collagen and Cytoskeletal Proteins
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Cells were lysed in RIPA buffer (150 μL/cm3) prior to SDS-PAGE electrophoresis. The proteins were transferred from the gel to methanol-activated PVDF membranes at 110 V for 2 h. The membranes were washed twice with 1X TBST and then incubated with 5% non-fat milk for 1 h at room temperature in order to block nonspecific binding. Next, they were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: anti-collagen I (ab138492, Abcam), anti-GAPDH (ab181602, Abcam), anti-alpha smooth muscle actin (ab124964, Abcam), anti-LAMB3 (TD2381S, Abmart), anti-RhoA (ab187027, abcam), anti-MYL9 (ab191393, Abcam) and anti-ROCK1 (ab134181, Abcam). After washing, the blotted membranes were then incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase. The protein bands were then visualized by using an enhanced chemiluminescence detection system (ECL Substrate Kit, Abcam).
4
Western Blot Analysis of Protein Signaling
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Protein samples were collected from cell lysates. Protein was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and then were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). At room temperature, these membranes were blocked in PBS containing 5% nonfat milk for 1 h, and then incubated with primary antibodies overnight at 4°C. The following were the primary antibodies in this research: anti-MEX3A (dilution: 1:500, Abcam, UK, ab79046), anti-RhoA (dilution: 1: 500, Abcam, UK, ab187027), anti-ROCK1 (dilution: 1:1000, Abcam, UK, ab134181), anti-LIMK1 (dilution: 1:500, Abcam, UK, ab81046), anti-β-actin (dilution: 1:1000, Abcam, UK, ab8226), anti-RhoA (phospho S188) (dilution: 1:1000, Abcam, UK, ab41435), anti-phospho-ROCK1 (Tyr913) (dilution: 1:1000, Invitrogen, USA, PA5-105054) and anti-phospho-LIMK1 (Thr508) (dilution: 1:1000, Invitrogen, USA, PA5-104925). After being washed with TBST, these membranes were incubated with secondary antibody at 25°C for 1 h. Protein blots were visualized using enhanced chemiluminescence (Millipore, USA) [14 ].
5
Protein Expression Analysis in BEAS-2B Cells
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To investigate the protein levels including ARHGEF12 (Rabbit anti-ARHGEF12 antibody, Affinity, DF4432), BCAT1 (Rabbit anti-BCAT1, ab197941, Abcam), RhoA (Rabbit Anti-RhoA), ROCK1 (RabbitAnti-ROCK1, ab134181, Abcam), PTEN (Rabbit Anti-PTEN, ab32199, Abcam), mTOR (Rabbit Anti-mTOR, ab134903, Abcam), and pS6K1 (Rabbit Anti-pS6K1, ab32529, Abcam), BEAS-2B cells treated with GS were then transfected with miRNA. After 48 h, Western Blot was employed to detect protein expression. SDS-PAGE gels electrophoresis was used to separate 20 μg total cell proteins, which were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk at 4 °C overnight and then incubated with respective primary antibodies at different dilutions (ARHGEF12, 1:1000; BCAT1, 1:500; RhoA, 1:3000; ROCK1, PTEN, mTOR, pS6K1, 1:2000; GAPDH, 1:4500) at RT for 4 h. Afterwards, the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:6000, Millipore, Billerica, MA) at RT for 1 hr. After TBST washing, immunoreactive signals were detected using an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Inc.) and recorded by an AutoChemi Imaging System (UVP LLC, CA, USA). The gray value of the bands was analyzed using MCID Elite software (InterFocus Imaging Ltd., Linton, UK).
6
Western Blot Analysis of Cellular Proteins
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Total protein samples were extracted from cell samples with RIPA buffer (CWBio, Beijing, China) and protein concentration was measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amount of protein sample was separated on 12% SDS-PAGE gels followed by transferred to nitrocellulose membranes. Then, the membranes were blocked in 5% non-fat milk dissolved in TBST solution and incubated with primary antibodies against p300 (Abcam, Cambridge, MA, USA; ab275378, 1:1000 diluted), RhoA (ab187027, 1:2000 diluted), ROCK (ab134181, 1:1000 diluted), NF-κB p65(ab207297, 1:1000 diluted), HDAC1 (ab109411, 1:4000 diluted), COL1A1 (ab270993, 1:3000 diluted), COL2A1 (ab34712, 1:3000 diluted), and GAPDH (ab8245, 1:5000 diluted) overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h. The protein bands were visualized using an enhanced chemiluminescence reagent (Pierce Biotech, Inc., Rockford, IL, USA).
7
Western Blot Analysis of ROCK1 and Sp1
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Samples were heated in SDS buffer, separated by SDS-PAGE and transferred to PVDF (polyvinylidene fluoride) membranes. Then, the membranes were blocked and separately probed with anti-ROCK1 (Abcam, Cambridge, MA, USA, ab134181), and anti-Sp1 (Abcam, ab13370) overnight. β-Actin (Santa Cruz, Santa Cruz, TX, USA, sc-130656) was used as a loading control. After washing, the membranes were incubated with secondary antibodies (Santa Cruz) and visualized using the ECL (enhance chemiluminescence) Western Blotting Detection System (Tiangen, Beijing, China).
8
Immunoblotting of Retinal Proteins
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The protein extracts of the HRVECs and zebrafish retinas were run on SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes, which were then incubated with primary antibodies against α2 integrin (ab133557), β1 integrin (ab179471), RhoA (ab86297), ROCK1 (ab134181; Abcam, Cambridge, UK), MYPT1 (D6C1), phosphomyosin phosphatase targeting subunit 1 (p-MYPT1; Thr696), or β-actin (D6A8; Signaling Technology, Danvers, MA, USA), followed by secondary antibody incubation. The membranes were incubated with the corresponding secondary antibodies (Bioworlde Technology Company, Nanjing, Jiangsu, China) the next day.
9
Protein Expression and Oxidative Stress Analysis
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Proteins were separated through 12%–15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with antibodies against ROCK1 (1:1000, ab134181, Abcam), cleaved caspase-3 (1:1000, 9661, Cell Signaling Technology), Phospho-Myosin phosphatase targeting Thr853 (p-MYPT1) (1:1000, 4563, Cell Signaling Technology), 4-Hydroxynonenal (4-HNE)-protein adducts (1:3000, ab46545, Abcam), Cytochrome c (1:1000, 24,840, Cell Signaling Technology), Cytochrome c oxidase subunit 4I1 (COX IV) (1:1000, 4850, Cell Signaling Technology), and β-actin (1:5000, 2D4H5, Proteintech). After washing, membranes were incubated with secondary antibodies. Bands were detected using chemiluminescence reagent. The intensity of the bands was quantified using ImageJ software.
10
Quantifying Subcellular Protein Localization
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