The renal cortical tissues of rats in different groups were respectively homogenized in lysis buffer and centrifuged. The supernatants were collected. Following quantification of protein concentrations, the tissue lysates (40 μg protein/lane) were separated by SDS-PAGE on a 7.5% polyacrylamide gel, followed by transferring onto nitrocellulose membranes (GE Healthcare, Beijing, China). Subsequently, the membranes were blocked in 5% skim dry milk in PBS and incubated with polyclonal rabbit anti-rat CTGF (1 : 200 dilution, Wuhan Boster Biological Technology, Ltd., Wuhan, China), anti-rat TGF-β1 (1 : 100 dilution, Wuhan Boster Biological Technology, Ltd., Wuhan, China), or anti-β-actin (1 : 1000 dilution, Wuhan Boster Biological Technology, Ltd., Wuhan, China) at 4°C overnight. The bound antibodies were detected with horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG and visualized using an enhanced chemiluminescence kit, according to the manufacturers' instruction (GE Healthcare, Beijing, China). The relative levels of target proteins to control β-actin were determined by densimetric scanning.
Anti β actin
Manufactured by Wuhan Boster Biological Technology
Sourced in China
Anti-β-actin is a primary antibody that recognizes the β-actin protein. β-actin is a highly conserved cytoskeletal protein found in all eukaryotic cells and is commonly used as a loading control in western blotting experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Anti pten, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti cav 1, by Santa Cruz Biotechnology (1 mentions)
Anti p mapk, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Anti pcna, by Wuhan Boster Biological Technology (1 mentions)
Enhanced chemiluminescence system kit, by Merck Group (1 mentions)
Ly294002, by Beyotime (1 mentions)
Bm0627, by Wuhan Boster Biological Technology (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti akt and anti pakt ser473, by Cell Signaling Technology (1 mentions)
Anti cd63, by Abclone (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
5 protocols using anti β actin
1
Western Blot Analysis of Renal CTGF and TGF-β1
2
Plasmid and Antibody Characterization
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The KCNA5 plasmid was from Dr Jie Zheng (University of California, Davis). The Cav-1 plasmid and siRNA plasmid specific for Cav-1 (target sequence Oligo 1, 5′-ACCTCATTAAGAGCTTCCTGATTGAGTCAAGAGCTCAATCAGGAAGCTCTTAATTT-3′, Oligo 2, 5′-CAAAAAATTAAGAGCTTCCTGATTGAGCTCTTGACTCAATCAGGAAGCTCTTAATG-3′) were obtained from the Cancer Center at Creighton University.
Anti-KCNA5 (rabbit polyclonal, 1:500; EMD Millipore, Billerica, MA, USA), anti-Cav-1 (mouse monoclonal, 1:1,000, Santa cruz biotechnology), anti-p-MAPK (mouse monoclonal, 1:1,000), anti-MAPK (rabbit polyclonal, 1:1,000), anti-p-AKT (rabbit monoclonal, 1:1,000) (all from Cell Signaling Technology, Danvers, MA, USA), anti-AKT (goat polyclonal, 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 1:500), anti-PCNA (mouse monoclonal, 1:500) and anti-β-actin (mouse monoclonal, 1:1,000) (all from Wuhan Boster Biological Technology, Ltd., Wuhan, China). HRP-conjugated goat anti-rabbit, anti-mouse or anti-goat specific secondary antibody (1:6,000; Zhongshan Golden Bridge Biotechnology, Beijing, China).
Anti-KCNA5 (rabbit polyclonal, 1:500; EMD Millipore, Billerica, MA, USA), anti-Cav-1 (mouse monoclonal, 1:1,000, Santa cruz biotechnology), anti-p-MAPK (mouse monoclonal, 1:1,000), anti-MAPK (rabbit polyclonal, 1:1,000), anti-p-AKT (rabbit monoclonal, 1:1,000) (all from Cell Signaling Technology, Danvers, MA, USA), anti-AKT (goat polyclonal, 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), 1:500), anti-PCNA (mouse monoclonal, 1:500) and anti-β-actin (mouse monoclonal, 1:1,000) (all from Wuhan Boster Biological Technology, Ltd., Wuhan, China). HRP-conjugated goat anti-rabbit, anti-mouse or anti-goat specific secondary antibody (1:6,000; Zhongshan Golden Bridge Biotechnology, Beijing, China).
3
Investigating PTEN/Akt Signaling in Angiogenesis
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The expression of PTEN was assessed by western blot analysis, and its expression in the samples was normalized to β-actin expression. Cells were lysed in RIPA buffer with freshly added protease inhibitor. Total lysates were separated on SDS-PAGE gels and transferred to polyvinylidene uoride (PVDF) membranes (Millipore). The immunoblots were blocked with 5% fat-free milk and they were incubated at 4˚C overnight with primary antibodies anti-PTEN (1:1000, Cell Signaling Technology), anti-β-actin (1:1000, BM0627; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Akt (Ser473) (1:1000, Cell Signaling Technology) phosphorylation were examined by using phospho-speci c antibodies. Total protein was determined using anti-Akt antibodies. After incubation with the secondary antibody, the membranes were visualized with an enhanced chemiluminescence system kit (Millipore, USA), according to the manufacturer's protocol. To explore whether miR-21 promote angiogenesis in tumor microenvironment by PTEN/Akt signaling pathway, the recipient cells were treated PI3K/Akt inhibitor LY294002 which was obtained from Beyotime Biotechnology (Nantong, China) with a nal concentration of 60μM.
4
Western Blot Analysis of Cellular Proteins
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor. After quantification by BCA kit (Beyotime Biotechnology), the denatured protein was separated on SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The immunoblots were blocked with 5% fat‐free milk and incubated at 4°C overnight with primary antibodies anti‐CD63 (1:1200, Abclone), anti‐PTEN (1:1000, Cell Signaling Technology), anti‐β‐actin (1:1000, Wuhan Boster Biological Technology, China), anti‐Akt and anti‐p‐Akt (Ser473) (1:1000, Cell Signaling Technology). After incubation with the secondary antibody, the bands were detected by enhanced chemiluminescence kit on in the Gel‐Pro Analyzer software.
5
Protein Quantification and Western Blot Analysis
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The cells were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) to obtain total proteins. The BCA method was used to determine the concentration of proteins. Following quantitation with a WBC Protein Quantitation kit (Thermo Fisher Scientific, Inc.), total proteins were separated via SDS-PAGE (12% gel) and transferred onto PVDF membranes (EMD Millipore). After 4 h of blocking with 5% skimmed milk at 4˚C, the primary antibodies, including anti-TP53INP1 (1:500; cat. no. A04229; Wuhan Boster Biological Technology, Ltd.) and anti-β-actin (dilution, 1:500; cat. no. BA2305; B Wuhan Boster Biological Technology, Ltd.), were incubated with the membranes at 4˚C overnight. Next, the membranes were incubated with an HRP-conjugated secondary antibody (1:5,000; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) at 37˚C for 2 h. In this analysis, β-actin was used as an internal control. Subsequently, an enhanced chemiluminescence BeyoECL Plus kit (Beyotime Institute of Biotechnology) was applied for protein band visualization. The protein bands were quantified using ImageJ Software v1.46 (National Institutes of Health).
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