Total protein was extracted from cultured cells or tissue using RIPA (CWBIO, Shanghai, China) and protease inhibitor (CWBIO, Shanghai, China). Protein concentration was determined using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were electrophoresed in 10% SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and sealed with 5% skimmed milk for 2 h at room temperature and incubated with anti-BDNF antibody(1:1000, Abcam, USA) or anti-PSD95 antibody(1:1000, Proteintech, Wuhan China) overnight at 4 °C. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected by an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). Image J software (National Institutes of Health, USA) was used to quantify the band densities.
Protease inhibitor
Manufactured by CWBIO
Sourced in China
Protease inhibitor is a type of lab equipment that functions to prevent the activity of proteases, which are enzymes responsible for breaking down proteins. It serves as an essential tool for researchers and scientists studying protein interactions, stability, and structure.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Radioimmunoprecipitation assay (ripa), by CWBIO (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Phosphatase inhibitor, by CWBIO (3 mentions)
Ripa buffer, by CWBIO (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg, by Boster Bio (2 mentions)
Cyclin b, by Santa Cruz Biotechnology (2 mentions)
Cyp11a1, by Abcam (2 mentions)
Membrane hrp goat anti mouse igg, by Boster Bio (2 mentions)
Rabbit anti goat igg secondary antibodies, by Boster Bio (2 mentions)
Chemiluminescence western blotting substrate, by Santa Cruz Biotechnology (2 mentions)
Cyp19a1, by Abcam (2 mentions)
Radioimmunoprecipitation assay (ripa), by Applygen (2 mentions)
Ripa lysis buffer, by CWBIO (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Anti bdnf antibody, by Abcam (1 mentions)
Thermo scientific pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l alexa fluor 488, by Abcam (1 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Abcam (1 mentions)
32 protocols using protease inhibitor
1
Quantifying Protein Expression in Cell Cultures
2
Protein Isolation and Western Blot Analysis
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The cell total protein was isolated using RIPA (Applygen Technologies Inc., Beijing, China). Protease inhibitor (CWBIO, Shanghai, China) was added into the RIPA at a ratio of 1:100. After adding RIPA to the cell culture plate, we collected the cells and centrifuged (12,000 r/min) the material at 4 °C for 10 min [26 ]. Protein concentrations were measured on a Thermo Scientific Pierce BCA protein assay kit (Thermo Fisher, Massachusetts, USA) with 1/4 volume of 5 × loading buffer added to the supernatant. A total of 20 μL of protein was blotted using 10% SDS-polyacrylamide gel, then transferred to a polyethylene difluoride (PVDF) membrane (CST, Boston, MA, USA).
After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies (1:1,000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1/SF-1 (Abcam, Cambridge, UK) and against cyclin B, cyclin D, cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, Wuhan, China) were diluted 1:3,000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software Image Lab™, (Bio-Rad, Berkeley, CA, USA).
After blocking with 5% defatted milk for 2 h, the membranes were incubated overnight at 4 °C with antibodies (1:1,000) against StAR, CYP19A1, CYP11A1, Mfn2, NR5A1/SF-1 (Abcam, Cambridge, UK) and against cyclin B, cyclin D, cyclin E, CDK4 (Santa Cruz, TX, USA). The membrane HRP goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG secondary antibodies (BOSTER, Wuhan, China) were diluted 1:3,000 according to the instructions and incubated for 1 h. Detection was performed using chemiluminescence Western blotting substrate (Santa Cruz, CA, USA) in Image Lab analysis software Image Lab™, (Bio-Rad, Berkeley, CA, USA).
3
Antibody and Reagent Procurement for Cell Signaling
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Rabbit monoclonal anti-IRF3, GAPDH, eGFP-Tag, HA-Tag and HRP-conjugated goat anti-rabbit IgG antibodies, and mouse monoclonal anti-Myc-Tag Flag-Tag antibody were purchased from Cell Signaling Technology (USA). The JetPRIME kit was purchased from Polyplus Transfection (France) and Double-Luciferase Reporter Assay Kit was purchased from TransGen (China). HRP-conjugated goat anti-mouse-IgG antibody, protease inhibitor, and phosphatase inhibitor were provided from CWBIO (China). The Pierce Crosslink Magnetic IP/Co-IP Kit was purchased from Thermo Scientific (USA). Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) were purchased from Abcam (USA). TRIM21 Polyclonal antibody was purchased from Proteintech (USA). MG132, chloroquine diphosphate (CQ), 3-methyladenine (3-MA) and Z-VAD-FMK were purchased from MedChemExpress (MCE) (USA).
4
Protein Expression Analysis by Western Blot
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The expression of proteins encoded by genes up- and downstream of miR-128-3p was detected by Western blot analysis. Cells were lysed with the NP40 Extraction Reagent (CWBio, Beijing, China) and supplemented with a protease inhibitor (CWBio, Beijing, China). The bicinchoninic acid assay was used to measure protein concentrations. Extracted proteins were mixed with loading buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to 0.2-μm nitrocellulose membranes. Primary antibodies (GFAP, 1:500; β III-tubulin, 1:400; NICD, 1:200; HES5, 1:500; CREB, 1:500, GAPDH, 1:5000; and histone, 1:5000) and horseradish peroxidase (HRP)-labeled secondary antibodies (1:10,000) were purchased from Abcam, Cambridge, MA, United States. Proteins were visualized with the Pierce ECL Western blotting substrate (CWBio, Beijing, China) for HRP. GAPDH and histone were used as internal controls.
5
Western Blot Analysis of Apoptosis Markers
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Cells were washed twice with pre-cooled PBS, mixed with RIPA lysate (Applygen, Beijing, China) and protease inhibitor (CWBIO, Beijing, China). Total proteins were isolated by utilizing a protein extraction reagent, denatured by boiling for 10 min, separated with SDS-PAGE, and transferred to PVDF membranes. Blocking of the membrane was performed with 5% non-fat dry milk and 0.2%Tween for 2 h at room temperature. Proteins were incubated with antibodies, including anti-Cleaved-Caspase3, anti-Cleaved-Caspase9, anti-BAX and anti-HSP90, overnight at 4 °C. Incubation with secondary antibody for 2 h was then conducted at room temperature. Finally, the bands were detected by ECL.
6
Western Blot Protein Analysis Protocol
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Total protein was obtained from cells and tissues in RIPA lysis buffer (Biyuntian, Shanghai, China) with protease inhibitor (CWBIO, Taizhou, Jiangsu, China) and then separated by 10% SDS-PAGE. Membranes were blocked with 5% milk and then incubated with the following primary antibodies (for all antibodies, see Supplementary Table S3 ), the concentrations were all 1:1000. Then, the membranes were incubated with the secondary antibody anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (CWBIO, Jiangsu, China), the concentrations were 1:8000. Imaged with a Sapphire Biomolecular Imager (Azure Biosystems, Dublin, OH, USA).
7
Protein Expression Analysis by Western Blot
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Cells were harvested using RIPA lysis buffer (CWBIO, Jiangsu, China) containing protease inhibitor (CWBIO, Jiangsu, China) and incubated on ice for 30 min. Subsequently, WB was performed using the method used in the previous study [24 (link)]. The membranes were incubated with the following primary antibodies: PCK2 (Shenggong, Shanghai, China), METTL3 (Shenggong, Shanghai, China), FTO (Fitzgerald, Acton, USA) and β-actin (Absin, Shanghai, China). Finally, the images were obtained with a Bio-Rad GelDoc system equipped with a Universal Hood III (Bio-Rad, California, USA), and the integrated optical density (IOD) was calculated using Gel-Pro Analyzer 4.0.0.4. Actin was used as an internal control.
8
Drosophila Schneider S2 Cell Culture
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Drosophila Schneider S2 cells, kindly provided by Prof. Jianguo He of Sun Yat-sen University, Guangzhou, China, were cultured at 27 °C for 48 h in Schneider’s Drosophila Medium (Cat# 21720-024, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Cat# 10091148, Gibco Life Technologies, Grand Island, NY, USA). Confluent cells were plated onto 6-well plates (Cat#712001, NEST Biotechnology, Shanghai, China) at 1.5 × 106 cells per well in 2 ml medium. At 60–80% confluence, cells were transfected with 2 μg of pIZ-PvHMC-Flag or the empty vector pIZ-V5/His using the FuGENE@ HD Transfection Reagent (Cat#E2311, Promega, Madison, WI, USA). At 36 h and 48 h post-transfection, cells were harvested, washed three times with pre-cooled PBS, and lysed with lysis buffer containing protease inhibitor (Cat# CW233, Cwbio, Beijing, China) plus 2× PMSF before being centrifuged at 20,000× g (4 °C, for 20 min). The obtained supernatant was mixed with 5× loading buffer and boiled for 10 min. Samples were used immediately or stored at −20 °C for later use.
9
Locust Brain Protein Analysis
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Locust brains (8–10 individuals/sample) were collected and homogenized in lysis buffer (CWBIO) containing protease inhibitor (CWBIO). The total protein content was determined using a bicinchoninic acid protein assay kit (Thermo Scientific). The extracts were reduced, denatured, and separated by gel electrophoresis on a 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated separately with specific antibodies against Piwi1, Piwi2, and Ago3 (mouse anti‐Piwi1 serum, 1:500; mouse anti‐Piwi2 serum, 1:500; and mouse anti‐Ago3 serum, 1:500, respectively), hnRNP F/H (Mouse monoclonal antibody, 1:2,000, Abcam), and U2AF65 (Mouse monoclonal antibody, 1:200, Santa Cruz Biotechnology). Tubulin was used as an endogenous control (rabbit polyclonal antibody, 1:5,000, CWBIO). Goat anti‐rabbit IgG was used as the secondary antibody (1:10,000, CWBIO). The intensities of the Western blot bands were quantified using densitometry with Quantity One software.
10
Viral and Bacterial Lung Quantification
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Mouse lungs were removed post-necropsy and placed in 2 mL EP tubes, after which 1 mL of PBS and 1% protease inhibitor (CW Biotech, Beijing, China) were added and the EP tubes were automatically ground in a high-throughput tissue grinder. The supernatant was collected after centrifugation at 10,000 g in 4°C for 15 min. The influenza virus titer in the lungs was measured using the fluorescence quantitative PCR method. The plate count method was used for quantitative determination of S. aureus. The lungs were triturated with a Polytron PT100 (Glen Mills, Clifton, NJ, USA) and the volume was adjusted to 1 mL with PBS to achieve the desired viral and bacterial titers.
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