Proteins were extracted and separated according to previously described methods. Membranes were incubated overnight at 4 °C with primary anti-RHOA, anti-RHOC, anti-RAC2, anti-RAB10, anti-RAB11B, anti-RAB11A, anti-RHOGDI, anti-PFN1, anti-LASP1, or anti-FOSL2 antibodies (Proteintech, Wuhan, China). Blots were visualized by exposure to X-ray films using an enhanced chemiluminescence detection system (EMD Millipore, Burlington, MA, USA). Blots were probed with anti-GAPDH antibody (Cell Signaling Technology, Danvers, MA, USA) as an endogenous control for equal loading.
Anti rhoa
Manufactured by Proteintech
Sourced in China, United States, United Kingdom
Anti-RhoA is a research-grade antibody designed to detect the RhoA protein. RhoA is a small GTPase that regulates various cellular processes. This antibody can be used in techniques such as Western blotting to identify and quantify RhoA expression levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Proteintech (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Anti mlc, by Proteintech (2 mentions)
Anti gapdh, by Yeasen (2 mentions)
Ab181858, by Proteintech (2 mentions)
Anti rap1, by Abcam (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence detection system, by Merck Group (1 mentions)
Anti rab11a, by Proteintech (1 mentions)
Anti pfn1, by Proteintech (1 mentions)
Anti lasp1, by Proteintech (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti gst, by Cell Signaling Technology (1 mentions)
Anti daam1, by Proteintech (1 mentions)
Anti ror2, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Anti p cofilin, by Abcam (1 mentions)
Drosha, by Abcam (1 mentions)
Anti hif1a, by Abcam (1 mentions)
9 protocols using anti rhoa
1
Western Blot Analysis of Cytoskeletal Proteins
2
Wnt5a Signaling Pathway Analysis
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KYSE410 and KYSE520 cells treated with CCG-1423 (Cat. S7719, Selleck Chemicals, Houston, TX, USA) or transfected with indicated constructs were washed with PBS and lysed with RIPA lysis buffer.19 (link) The lysates were then clarified by centrifugation at 12,000xg for 20 mins at 4°C. The protein extracts were separated by 8% or 10% SDS-PAGE. The following antibodies were used: anti-Wnt5a (1:1,000 dilution, Cat. sc-365370, Santa Cruz Biotechnology), anti-β-actin (1:5,000 dilution, Cat. 60008-1-Ig, ProteinTech Group, Wuhan, China), anti-ROR1 (1:1,000 dilution, Cat. 20629-1-AP, ProteinTech), anti-ROR2 (1:1,000 dilution, Cat. 88639, Cell Signalling Technology, Danvers, MA, USA), anti-flag (1:1,000 dilution, Cat. 14793, Cell Signalling Technology), anti-GST (1:1,000 dilution, Cat. 2624, Cell Signalling Technology), anti-RhoA (1:1,000 dilution, Cat. 10749-1-AP, ProteinTech), and anti-DAAM1 (1:1,000 dilution, Cat. sc-100942, Santa Cruz Biotechnology) antibodies. Horseradish peroxidase-conjugated antibodies and Pierce™ ECL western blotting substrate (Cat. 32209, Thermo Fisher Scientific, Waltham, MA, USA) were used to exposure protein bands. The pictures were taken by Tanon 5200 Multi Imaging Workstation (Tanon, Shanghai, China) and the densitometry of immunoblots was analyzed by ImageCal software (Tanon).
3
Immunohistochemical Staining of Key Markers
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Immunohistochemical staining was performed as described previously [23 (link)]. Briefly, the deparaffinized tissue sections were heated for antigen retrieval, quenched for endogenous peroxidase activity and blocked with goat serum; the primary antibodies against Drosha (1:150; Abcam), anti-ABHD16A (BAT5) (1:200; GeneTex), anti-GPR34 (1:150; Abcam), anti-HIF1A (1:150; Abcam), anti-RhoA (1:150; Proteintech), anti-p-LIMK1 (1:150; Abcam), anti-p-cofilin (1:150; Abcam), and secondary antibody (1:100; ZSBIO, Beijing, China) were used. After staining with diaminobenzidine and hematoxylin, the images were captured.
Immunoreactivity was evaluated independently by two researchers who were blinded to patient outcome. Scoring criteria for staining intensity were as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). The staining extent score was as follows: 0 (<10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The final expression score, ranging from 0 to 12, was calculated as intensity scorexextent score. Total scores of 4 or lower were categorized as the low expression group, and the rest as the high expression group.
Immunoreactivity was evaluated independently by two researchers who were blinded to patient outcome. Scoring criteria for staining intensity were as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). The staining extent score was as follows: 0 (<10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The final expression score, ranging from 0 to 12, was calculated as intensity scorexextent score. Total scores of 4 or lower were categorized as the low expression group, and the rest as the high expression group.
4
Protein Expression Analysis in Cells
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Cells or tissues were harvested and lysed by RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Solarbio Life Science, China). The concentrations of total protein were detected via a BCA Protein Assay Kit (Solarbio Life Science, China) and equal amounts of protein were loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then proteins were transferred onto Nitrocellulose (NC) membrane (Pall Corporation, USA). Later, NC membranes were incubated with different primary antibodies (anti-α-SMA, anti-CD31, anti-NSE, anti-GAP43, anti-S100β, anti-RhoA and anti-Rock1, ProteinTech Group, Inc) at 4 °C overnight and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The signals were detected with the BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).
5
NPHP1 Knockout Mouse Model
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NPHP1KO C57BL/6J mice were generated by our group by deleting exons 2 to 20 of NPHP1 using the CRISPR/Cas9 technique [52 (link)]. All animal experiments were performed according to the ARRIVE1 guidelines, and protocols were approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University.
The following primary antibodies were used: anti-NPHP1 (Sigma Aldrich, Sab2104055, St. Louis, MO, USA), anti-GEF-H1 (CST, #4067, Danvers, MA, USA), anti-GAPDH (proteintech, 10494-1-AP, Rosemont, IL, USA), anti-RhoA (Proteintech, 10749-1-AP, Rosemont, IL, USA), anti-MLC2 (CST, #3672, Danvers, MA, USA), anti-pMLC2 (CST, #3671, Danvers, MA, USA), anti-F4/80 (Abcam, ab300421, Waltham, MA, USA), anti-α-SMA (CST, 19245S, Danvers, MA, USA), anti-E-cadherin (Santa Cruz, SC-1500, Dallas, TX, USA), and anti-α-tubulin (Immunoway, YM3035, Plano, TX, USA). Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were also used.
The following primary antibodies were used: anti-NPHP1 (Sigma Aldrich, Sab2104055, St. Louis, MO, USA), anti-GEF-H1 (CST, #4067, Danvers, MA, USA), anti-GAPDH (proteintech, 10494-1-AP, Rosemont, IL, USA), anti-RhoA (Proteintech, 10749-1-AP, Rosemont, IL, USA), anti-MLC2 (CST, #3672, Danvers, MA, USA), anti-pMLC2 (CST, #3671, Danvers, MA, USA), anti-F4/80 (Abcam, ab300421, Waltham, MA, USA), anti-α-SMA (CST, 19245S, Danvers, MA, USA), anti-E-cadherin (Santa Cruz, SC-1500, Dallas, TX, USA), and anti-α-tubulin (Immunoway, YM3035, Plano, TX, USA). Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) were also used.
6
Protein Expression Analysis by Western Blot
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Protein extracts from cells were prepared using 2% SDS lysis buffer including protein phosphatase inhibitor (Thermo Scientific™, #78,440). Total protein (20 μg) was subjected to 10% (v/v) SDS-PAGE gels and transferred to nitrocellulose filter membrane (Pall, #P-N66485). After blocking with 5% bovine serum albumin (BSA) for 2 h at 37 ℃, the membranes of proteins with different molecular weight (KDa) were cut according to the location of the markers (GenStar, M222) with enough spaces left at the edges of the first and the last sample lanes, then the cropped membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies anti-KRT7 (1:1000; Proteintech, #17,513–1-AP), anti-E-cadherin (1:5000; Proteintech, #20,874–1-AP), anti-P-cadherin (1:1000; Proteintech, #13,773–1-AP), anti-RHOA (1:1000; Proteintech, #10,749–1-AP), anti-Phospho-MLC2 (1:1000; Cell Signaling Technology, #3671), anti-β-Actin (1:20,000; Proteintech, #66,009–1-lg) and anti-GAPDH (1:50,000; Proteintech, #60,004–1-lg) were used and then incubated with HRP-conjugated secondary antibodies (1:5000; Proteintech, #SA00001-1 and # SA00001-2) at room temperature for 1 h and imaged through ECL kit (Beyotime, #P0018AM).
7
RhoA Activity Assay Protocol
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The RhoA activity assays were performed according to the manufacturer's protocol (Upstate Cell Signaling Solutions, Lake Placid, NY). Active Rho (Rho-GTP) was isolated with a GST fusion protein containing the RhoA binding domain of rhotekin (GST-rhotekin RBD). The cells were washed with ice-cold PBS and lysed on ice in cell lysis buffer. Following determining the protein concentrations using the method of Bradford, 250 µg total protein per sample were incubated with GST-rhotekin RBD immobilized on glutathione-Sepharose 4B beads (GE Healthcare Life Sciences) at 4°C for 60 min. The beads were pelleted and washed three times with lysis buffer. Bound GTP-RhoA was eluted from the beads with SDS gel sample buffer, boiled for 5 min and separated by 12% SDS-PAGE. The active Rho proteins were detected by immunoblotting using anti-RhoA (ProteinTech Group, Inc.) antibody. Signals were quantified using ImageJ software 2× (National Institutes of Health, Bethesda, MD, USA).
8
Quantifying Cell Signaling Proteins
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The relative levels of Rap1, RhoA, Ve-cadherin, MLC expression and MLC phosphorylation in individual groups of cells were quanti ed by Western blot. Brie y, the different groups of cells were harvested and lyzed in lysis buffer, followed by centrifuged. After quanti cation of protein concentrations, the cell lysates (50 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene di uoride (PVDF) membranes. After being blocked, the membranes were probed with primary antibodies including anti-Rap1 (Abcam, ab181858, UK), anti-RhoA, (Proteintech, 10749-1-AP, USA), anti-MLC (Proteintech, 10906-1-AP), anti-p-MLC (Cell Singnaling Technology, 3671, USA), anti-Ve-cadherin (Cell Singnaling Technology, 2500) and anti-GAPDH (Yeasen Biotech, 33106ES60, China). After being washed, the bound antibodies were detected with HRPconjugated second antibodies and visualized with enhanced chemiluminescent reagents. The relative levels of each target to the control protein were quanti ed by densitometric analysis using Image-Pro Plus software (Media Cybernetics).
9
Quantification of Cell Signaling Proteins
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The relative levels of Rap1, RhoA, Ve-cadherin, MLC expression and MLC phosphorylation in individual groups of cells were quantified by Western blot. Briefly, the different groups of cells were harvested and lyzed in lysis buffer, followed by centrifuged. After quantification of protein concentrations, the cell lysates (50 µg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked, the membranes were probed with primary antibodies including anti-Rap1 (Abcam, ab181858, UK), anti-RhoA, (Proteintech, 10749-1-AP, USA), anti-MLC (Proteintech, 10906-1-AP), anti-p-MLC (Cell Singnaling Technology, 3671, USA), anti-Vecadherin (Cell Singnaling Technology, 2500) and anti-GAPDH (Yeasen Biotech, 33106ES60, China). After being washed, the bound antibodies were detected with HRP-conjugated second antibodies and visualized with enhanced chemiluminescent reagents. The relative levels of each target to the control protein were quantified by densitometric analysis using Image-Pro Plus software (Media Cybernetics).
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