Commercially available primary antibodies specific for the following proteins were used: CD74 (Sigma-Aldrich, SAB1402715); CD44 (Santa Cruz, sc-7297); p-CFL1 (Sigma-Aldrich, SAB4300115); ROCK1 (Proteintech, 21850-1-AP); RHOA (CST, #2117P); RAC1 (CST, #2465P); and CDC42 (CST, #2466P). TRITC-phalloidin (P1951) was purchased from Sigma-Aldrich, and the Transcriptor First Strand cDNA Synthesis Kit (04897030001) was obtained from Roche.
Rock1
Manufactured by Proteintech
Sourced in China, United States
ROCK1 is a protein kinase that plays a crucial role in regulating cell cytoskeleton and cell contractility. It is involved in various cellular processes, including cell migration, cell adhesion, and smooth muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
Cofilin, by Proteintech (2 mentions)
P limk2, by Abcam (2 mentions)
P cofilin, by Abcam (2 mentions)
Synaptopodin, by Santa Cruz Biotechnology (2 mentions)
Ecl system, by Share-Bio (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
P cfl1, by Merck Group (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
C ebpα, by Proteintech (1 mentions)
Fabp4, by Proteintech (1 mentions)
Rock2, by Proteintech (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Erk1 2, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
10 protocols using rock1
1
Antibody-based Investigation of Cytoskeletal Regulators
2
Western Blot Analysis of Adipogenic Markers
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Cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Beyotime, Haimen, China). Total and nuclear proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). Protein samples (50 μg) were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBST containing 5% fat-free milk at room temperature for 1 h. After washing with TBST, the membranes were probed with primary antibodies against C/EBPα, FABP4, PPARγ, RhoA, ROCK1, ROCK2, ERK1/2, β-actin, PCNA (all Proteintech, Wuhan, China), and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed in TBST and incubated with secondary antibody (Proteintech, Wuhan, China). The signals were developed by ECL reagents (Beyotime, Haimen, China).
3
Western Blot Analysis of Penile Tissue Proteins
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As in our previous study,17 (link)18 (link) penile tissues from each group were homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Concentrations of soluble proteins were measured using the bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Protein samples (40 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking in Tris buffered saline-Tween (TBST) with 5% bovine serum albumin, membranes were incubated overnight at 4°C with primary antibodies against: hKLK1 (1:5000, Sigma-Aldrich, St. Louis, MO, USA), rKLK1 (1:1000, Sigma-Aldrich, St. Louis, MO, USA), α-SMA (1:1000, Abcam), TGF-β1 (1:1000, Abcam), RhoA (1:1000, Proteintech), ROCK1 (1:1000, Proteintech), p-MYPT1 (1:1000, Millipore, Billerica, MA, USA), LIMK2 (1:1000, Abcam, Cambridge, MA, USA), p-LIMK2 (T505, 1:1000, Abcam), cofilin (1:1000, Proteintech, Wuhan, China), and p-cofilin (1:1000, Abcam). Washed membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Finally, bands were developed with an enhanced chemiluminescence detection system.
4
Protein Expression Analysis of Prostate Tissue
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The prostatic tissue was ground in liquid nitrogen and the total protein was extracted using a protein extraction kit. Equal amounts (40 μg/lane) of proteins were separated by SDS–polyacrylamide gel electrophoresis; the resultant bands were transferred onto PVDF membranes. After being blocked in 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with antibodies against TGF-β1 (1:500; Proteintech Group), RhoA (1:5000; Proteintech Group), ROCK1 (1:500; Proteintech Group), and β-actin (1:1000; Affinity Biosciences) overnight at 4 °C. The membranes were washed thrice in TBST for 30 min, and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Affinity Biosciences) for 1 h followed, by a further washing step for 30 min. Finally, the bands were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific). The data were normalized using β actin as an internal control. All samples were analyzed independently via three repetitions and the mean values were determined.
5
Analysis of RhoA/ROCK Signaling Pathway
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Total protein samples were extracted from cells after incubating with valsartan, tetrandrine and Y-27632 for 48 h or with U73122 for 10 min. RIPA lysis buffer (Beyotime) with 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime) was used for cell lysis. The supernatant was transferred to a centrifuge tube for vortexing. Protein lysates were centrifuged at 4°C, 12,000 × g for 10 min. Protein samples were subjected to 10% SDS-PAGE for separation and then transferred onto polyvinylidene difluoride (PVDF) membranes according to standards methods. PVDF membranes were blocked with skim milk powder, and incubated with primary antibodies. against TRPC6 (1: 1000, Santa Cruz), Synaptopodin (1: 100; Santa Cruz), Nephrin (1: 100; Proteintech), ROCK1 (1: 1000; Proteintech), anti-RhoA antibody (1: 1000; Proteintech, Wuhan, China), and β-actin (1: 1000; Santa Cruz) at 4°C overnight. Activated RhoA was determined by measuring membrane-bound RhoA (GTP-RhoA) using the GST rhotekin-Rho binding domain that specifically pulls down activated RhoA, Secondary antibodies were peroxidase AffiniPure donkey anti-mouse or goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The blots were developed using ECL system (Share-bio, Shanghai, China) and analyzed using the TanonImage software (Tanon, Shanghai, China).
6
Retinal Immunohistochemistry Analysis
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After removal of the eye and fixation, the retinal tissue was sectioned with GLP-1R (Novus, Colorado, USA, Cat. No. NLS1205, 1 : 50), ROCK1, MMP-9, VEGF (Proteintech, Wuhan, China, Cat. No. 21850-1-AP, 1 : 2000), p-MLC (Bioss, Beijing, China, Cat. No. 10375-2-AP, 1 : 2000, Cat. No. 19003-1-AP, 1 : 2000), p-MLC (Bioss, Beijing, China, Cat. No. bs-4060R, 1 : 2000) Occludin (Signalway Antibody, Nanjing, China, Cat. No. 29275, 1 : 2000), and ZO-1 primary antibody (Biorbyt, Cambridge, UK, Cat. No. orb11587, 1 : 2000) were reacted overnight at 4°C, followed by goat anti-rabbit IgG (Signalway Antibody, Nanjing, China, Cat. No. L3012, 1 : 5000) secondary antibody for 60 min, visualized with DAB, and restained with hematoxylin and eosin (H&E). Finally, the films were sealed and photographed and observed with an Olympus microscope. The grayscale values of the positive colors of the above indicators for each group of three sections were analyzed using the ImageJ 18.0 mean positive staining area percentage method, which is the %Area measured.
7
Comprehensive Protein Analysis Protocol
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Western blot analysis was performed using standard methods. The antibodies used were as follows: RhoGDIα (1:1000; Abcam, USA), P-LIMK (1:1000; CST, USA), LIMK (1:1000; Proteintech, USA), P-cofilin (1:1000; CST, USA), cofilin (1:1000; Proteintech, USA), P53 (1:1000; CST, USA), Rac1 (1:1000; Proteintech, USA), Cdc42 (1:1000; Proteintech, USA), Pak4 (1:1000; Proteintech, USA), Rock1 (1:1000; Proteintech, USA), RhoA (1:1000; Affinity, China) and GAPDH (1:10000; Proteintech, USA). Rac1 and RhoA activity was detected by the Rac1/RhoA Pull-Down Activation Assay Biochem Kit (Cytoskeleton, USA). The Western blot bands were quantified by ImageJ software and normalized to GAPDH.
8
Western Blot Analysis of Signaling Pathways
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Equal amounts of protein were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and incubated at 4 °C overnight with antibodies against GPR34 (1:1500), phosphorylated (p)-TGF-β (1:1000), p-Smad2 (1:1500), p-Smad3 (1:2500), Smad2/3 (1:1000 (all from Affinity), CDK2 (1:2500), CDK6 (1:2200), cyclin E (1:1500), MMP-2 (1:1500), MMP-7 (1:1000), ROCK1 (1:3000), E-cadherin (1:1500), N-cadherin (1:1500), Snail (1:1500), vimentin (1:2000), and GAPDH (1:2500 [all from Proteintech, Wuhan, China]). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:2500, Affinity) at 37 °C for 3 h. Protein bands were visualized using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific). GAPDH served as a loading control.
9
Western Blot Analysis of Kidney Protein Markers
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Total protein was extracted from MPC5 podocytes and kidney tissues of rats. For sufficient cell lysis, RIPA lysis buffer (Beyotime, China) was added, and then, the supernatant was extracted after centrifugation at 12,000 rpm for 10 min. Protein samples were separated with 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with skim milk powder and then incubated with primary antibodies to anti-TRPC6 (1 : 1,000; Santa Cruz, USA), RhoA (1 : 1,000; Proteintech, Wuhan, China), ROCK1 (1 : 3,000; Proteintech), synaptopodin (1 : 1,000; Santa Cruz), and GAPDH (1 : 10,000; Abcam, UK). Membranes were then incubated with donkey anti-mouse IgG (H+L; Jackson ImmunoResearch Laboratories, USA) secondary antibody. ECL system (Sharebio, China) was used for band detection and protein bands were analyzed by the Tanon Image software (Tanon, China).
10
Penile Tissue Histology and Immunohistochemistry
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Specimens of penile tissue were cut in a transverse direction (thickness, 5 μm). The sections were deparaffinized with xylene and rehydrated in a graded ethanol series to distilled water. All of the sections were stained with Masson's trichrome staining to examine smooth muscle and connective tissue histology.
For immunohistochemical examination, sections were incubated overnight at 4°C with antibodies against: RhoA (1:50 dilution; Proteintech, Wuhan, China), ROCK1 (1:50; Proteintech, Wuhan, China), α-smooth muscle actin (α-SMA; 1:100; Abcam, Cambridge, MA, USA), transforming growth factor-β1 (TGF-β1; 1:100, Abcam, Cambridge, MA, USA), p-LIMK2 (1:100; Abcam, Cambridge, MA, USA), and p-cofilin (1:100; Abcam, Cambridge, MA, USA). After being washed, the sections were incubated with a biotinylated secondary antibody. Finally, antigen-antibody reactions were developed by diaminobenzidine. Semiquantitative analysis was performed to evaluate intensity using Image-Pro plus software (Media Cybernetics, Silver Spring, MD, USA).
For immunohistochemical examination, sections were incubated overnight at 4°C with antibodies against: RhoA (1:50 dilution; Proteintech, Wuhan, China), ROCK1 (1:50; Proteintech, Wuhan, China), α-smooth muscle actin (α-SMA; 1:100; Abcam, Cambridge, MA, USA), transforming growth factor-β1 (TGF-β1; 1:100, Abcam, Cambridge, MA, USA), p-LIMK2 (1:100; Abcam, Cambridge, MA, USA), and p-cofilin (1:100; Abcam, Cambridge, MA, USA). After being washed, the sections were incubated with a biotinylated secondary antibody. Finally, antigen-antibody reactions were developed by diaminobenzidine. Semiquantitative analysis was performed to evaluate intensity using Image-Pro plus software (Media Cybernetics, Silver Spring, MD, USA).
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