Proteins from cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO, China) with protease and phosphatase inhibitors (CWBIO, China). Identical quantities of proteins were electrophoresed by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% non-fat milk for 1 h, and incubated with primary antibodies specific for Bax, Bcl-2, Caspase-3 (#2772, #2872, and #9662) (1 : 1000, Cell Signaling Technology, USA), and β-actin (66009-1-Ig, 1 : 5000, Proteintech, USA) at 4°C overnight, followed by incubation with anti-rabbit/mouse HRP-conjugated secondary antibodies (cw0103s, cw0102s) (1 : 2000, CWBIO, China) at room temperature for 1 h. Signals were detected by Immobilon ECL substrate (Millipore, Germany).
Protease and phosphatase inhibitor
Manufactured by CWBIO
Sourced in China
Protease and phosphatase inhibitors are laboratory reagents used to prevent the degradation of proteins and phosphorylated molecules during sample preparation and analysis. These inhibitors work by interfering with the activity of enzymes that break down these biomolecules, helping to preserve their native structure and function for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by CWBIO (4 mentions)
Ripa buffer, by CWBIO (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Immobilon ecl substrate, by Merck Group (3 mentions)
Western lightning plus ecl kit, by PerkinElmer (3 mentions)
Gapdh, by Abcam (2 mentions)
Bca protein assay kit, by CWBIO (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti mouse igg antibody, by Southern Biotech (2 mentions)
Hrp conjugated anti rabbit igg, by Beyotime (2 mentions)
β actin 66009 1 ig, by Proteintech (1 mentions)
Twist, by Abcam (1 mentions)
Tsg101, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Bicinchoninic acid assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Klf10, by Santa Cruz Biotechnology (1 mentions)
19 protocols using protease and phosphatase inhibitor
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of EV Markers
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Cells or EVs were lysed in RIPA buffer (CWBIO, Beijing, China) with protease and phosphatase inhibitors (CWBIO). Identical quantities of proteins were electrophoresed by SDS‐PAGE, transferred onto PVDF membranes and incubated with primary antibodies specific for TSG101 (Abcam, Shanghai, China), CD63 (Abcam), TET1 (Abcam), Twist (Abcam) and GAPDH (Abcam) at 4°C overnight, followed by incubation with appropriate HRP‐conjugated secondary antibodies at room temperature for 1 hour. Signals were detected by Immobilon ECL substrate (Millipore, Germany), and the images were acquired using an Optimax X‐ray Film Processor (Protec, Germany).
3
Western Blotting Analysis of Skin and Cell Lysates
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Western blotting was performed according to a previously reported method (Ma et al., 2019 (link)). Mouse dorsal skin tissue or cultured DPCs were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (CWBIO, Jiangsu, China) to release the proteins. The latter were quantified with a bicinchoninic acid assay kit (Beyotime Biotechnology Inc. Shanghai, China) according to the manufacturer’s instructions. Equal amounts of protein lysates were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel electrophoresis. For specific antibody hybridization detection, the separated proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore Corporation, Billerica, MA, Unites States) which were then blocked with 5% (v/v) non-fat milk for 1 h and incubated with primary antibodies at 4°C overnight. The following day, the membranes were washed thrice with 0.1% (v/v) Tris-buffered saline with Tween-20. The membranes were then separately incubated with the corresponding secondary antibodies, subjected to an enhanced chemiluminescence kit, and observed under ChemiScope 6000 Series Chemiluminescence Imaging System (Clinx Science Instrument Co. Ltd. Shanghai, China) according to the manufacturer’s instructions.
4
Western Blot Analysis of EMT Markers
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Cells were collected and lysed with RIPA lysis buffer (Beyotime, Nanjing, China) with protease and phosphatase inhibitors added (CWBio, Beijing, China). Protein samples were separated by SDS-polyacrylamide-gel electrophoresis and transferred to polyvinylidene fluoride membranes. Then the membranes were incubated with the following primary antibodies at 4°C for 12 h: KLF10 (Santa Cruz Biotechnology, 1:1,000), Slug (Cell Signaling Technology, 1:1,000), E-cadherin (Cell Signaling Technology, 1:1,000), and GAPDH (Abcam, 1:5,000). Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. The protein-band signal was detected with the ECL Detection Kit (Millipore, Darmstadt, Germany) and visualized using an Optimax X-ray Film Processor (Protec, Oberstenfeld, Germany).
5
Quantifying Proteins in Tissue Samples
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Total proteins in human BC tissue samples, animal tissues and cells were extracted by RIPA lysis buffer with protease and phosphatase inhibitors (CwBio, Beijing) and subsequently quantified by the BCA method. For immunoblotting, equal amounts of proteins were electrophoresed on 8-10% SDS‒PAGE gels and subsequently transferred to PVDF membranes. After blocked the membranes with 5% BSA or 5% defatted milk for 1 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C. After being washed with TBST, membranes were incubated with commensurable secondary antibodies. Finally, the membrane was cleaned again with TBST and then exposed in Tanon 5200 (Shanghai, China) by dropping ECL regents on it. Data were analyzed by Image J V1.8.0. The antibodies used are shown in Supplementary Table 1.
6
Tissue Lysis and Protein Extraction
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The posterior pole tissues of eye balls were ground into powder in liquid nitrogen. Then, the tissue powder was lysed with 200 µl of neutral RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with sodium orthovanadate, sodium fluoride, EDTA, and leupeptin, CWBIO, Beijing, China) supplemented with protease and phosphatase inhibitors (CWBIO, Beijing, China). The lysate was subjected to ultrasonication for 1 min, and the resultant solution was centrifuged at 12,000 rpm/min at 4 °C for 20 min. The supernatant was aliquoted, one aliquot was for measuring the total protein concentration, and the others were stored at -80 °C for further usage.
7
Western Blot Analysis of AGTR1 Protein
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (all from CWBio). The concentration of protein was determined using a BCA Protein Assay kit. The same amount of total protein (40 µg protein per lane) was used for 10% SDS-PAGE. The resolved proteins were transferred to polyvinylidene fluoride membranes. The membrane was blocked with 5% BSA (Beyotime Institute of Biotechnology) for 1 h at room temperature, and incubated with antibodies to GAPDH (1:1,000 dilution; product code ab181602; Abcam) and AGTR1 (1:1,000; product code ab124505; Abcam) overnight at 4°C. This was followed by exposure to an appropriate secondary antibody conjugated with horseradish peroxidase at room temperature for 1 h. The secondary antibody used was as follows: HRP-labeled goat anti-rabbit IgG (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology). Immobilon ECL substrate (EMD Millipore) was used to generate signals, which were detected using the Optimax X-ray Film Processor (Protec GmbH & Co. KG). The protein bands were analyzed using ImageJ software (version 1.48; National Institutes of Health).
8
Western Blot Analysis of Adenovirus Proteins
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LMH cells infected with DAdV-3 or transfected with pcDNA3.1-Fiber-2 were washed and lysed with RIPA buffer (CWbio, Beijing, China) containing protease and phosphatase inhibitors (CST, MA, USA). The lysates mixed with loading buffer were boiled for 10 min and centrifuged for 3 min. After SDS-PAGE, the denatured proteins were transferred onto nitrocellulose membrane (GE Healthcare Life sciences, Freiburg, Germany). After blocked with 5% skim milk in PBST for 2 h at room temperature (RT), the membrane was incubated with the corresponding mAbs diluted with 5% skim milk in PBST for 2 h at RT. After three washes with PBST, the membrane was incubated with HRP-conjugated goat anti-mouse IgG diluted with 5% skim milk in PBST for 1 h. After another three washes, the membrane was developed with chemiluminescent reagents and imaged with an automatic imaging system (Tanon 5200).
9
Protein Expression Analysis by Western Blot
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Cells were harvested and lysed on ice in RIPA lysis buffer (CWBIO, China) containing protease and phosphatase inhibitors (CWBIO, China), followed by centrifugation at 14,000 rpm for 15 min at 4 °C. Bicinchoninic acid (BCA) protein assay kit (CWBIO, China) was used to quantify protein concentrations. Proteins from each sample were separated by SDS-PAGE gels, transferred to PVDF membranes, and then blocked with 5% BSA. The membranes were incubated overnight at 4°C with primary antibodies including AHNAK2 (1:1000, Proteintech, #17682-1-AP), c-MET (1:1000, #ab216574, Abcam), p-MET (Tyr1234/1235) (1:1000, #3077S, CST), Akt (1:1000, #4691, CST), p-Akt (Ser473) (1:2000, #4060, CST) and GAPDH (1:10,000, #ab181602, Abcam). The protein strips were then washed three times with 1×TBST and incubated with HRP-conjugated secondary antibodies at room temperature for an hour. Bands were detected with ECL detection system (Millipore, Germany) and captured using SmartChemi 910 plus (Sage, Beijing).
10
Protein Extraction and Western Blot
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Protein from cells and tissues was extracted using radioimmunoprecipitation assay (RIPA) buffer (Millipore, MA, and United States) with protease and phosphatase inhibitors (Cwbio, Beijing, and China) on ice for 30 min. The nuclear fraction was acquired through nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, and China) according to the manufacturer’s instruction. Ultrasonic homogenizers were used to disrupt the RIPA buffer-extracted lysates. Later, the supernatants were harvested by centrifugation for 25 min at 12000 rpm and their concentration was measured by a BCA protein assay kit (Cwbio, Beijing, and China). 4%–20% SDS-PAGE gel (SurePAGE™, GenScript, Nanjing, and China) was used to separate the proteins, which were then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA, and United States). Blocking was performed with 5% BSA in TBST for 1 h, followed by incubation with indicated primary antibodies at 4°C overnight and secondary antibodies for 1 h at room temperature with mild shaking. Protein bands were visualized using the ECL system (ZEN-BIOSCIENCE, Chengdu, and China) and all images were analyzed with ImageJ software. The antibody information is listed in Supplementary Table S2 .
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