Chicken lung tissues from each group was immersed in protein lysate and centrifuged, and supernatant was removed. The protein concentration was measured using the BCA detection kit. The same amount of protein was subjected to electrophoresis on polyacrylamide gel and transferred to a PVDF membrane using wet transformation method (110 V, 90 min). The membrane was blocked with 5% fetal bovine serum and incubated with primary NLRP3 polyclonal antibody (1:400, WanleiBiotechnology Co., Ltd., Shenyang, China), NF-κB p65 polyclonal antibody (1:1,000, bs-0465R, Bioss, Beijing, China), p-NF-κB p65 polyclonal antibody (1:1,000, bs-0982R, Bioss, Beijing, China), and β-actin polyclonal antibody (1:5,000, bs-0061R, Bioss, Beijing, China) overnight at 4°C. Further, the membrane was incubated with antirabbit IgG peroxidases (bs-0061R, Bioss, Beijing, China) for 1 h at room temperature. The bands were visualized and imaged using ECL (Biosharp Life Sciences, Hefei, China) and gel imaging system (tanon-5200, Tianneng Technology Co., Ltd., Shanghai, China). Image J software was used for the analysis of gray values.
Bs 0061r
Manufactured by Bioss Antibodies
Sourced in China, United States, United Kingdom
The Bs-0061R is a high-quality lab equipment product manufactured by Bioss Antibodies. It is designed for use in various research and laboratory applications. The core function of the Bs-0061R is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Ripa buffer, by Beyotime (6 mentions)
Anti β actin, by Bioss Antibodies (6 mentions)
Bs 0295g hrp, by Bioss Antibodies (4 mentions)
β actin, by Bioss Antibodies (3 mentions)
Anti cleaved caspase 3, by Bioss Antibodies (3 mentions)
Bs 0295g, by Bioss Antibodies (3 mentions)
Bca kit, by Beyotime (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Hrp conjugated anti rabbit igg antibody, by GeneTex (3 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Bca protein kit, by Beyotime (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Ab209845, by Abcam (3 mentions)
Bs 0465r, by Bioss Antibodies (2 mentions)
Bs 0982r, by Bioss Antibodies (2 mentions)
Goat anti rabbit igg, by Abcam (2 mentions)
Beclin1, by Bioss Antibodies (2 mentions)
Bs 40295g, by Bioss Antibodies (2 mentions)
63 protocols using bs 0061r
1
Quantifying NLRP3 and NF-κB Signaling
2
Intracerebral Hemorrhage Rat Model Protocol
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We selected the striatum in brain tissue for sampling. The striatum is the prone site of intracerebral hemorrhage. We used a stereotactic instrument to accurately inject autologous blood into the rat brain. We take brain tissue and preserve the tissue with a thickness of about 6 mm centered on the injection point. Brain tissues were homogenized in lysis buffer and centrifuged, and the supernatant (50 g total protein) was fractionated by SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride membrane and blocked in 5% skim milk for one hour at room temperature. Membranes were incubated at 4°C overnight with rabbit primary antibodies against Beclin1 (Bioss, China), Parkin (Bioss, China), PINK1 (CST, USA), BNIP3L (CST, USA), or Caspase-9 (bs-0049R, Bioss, China). Blots were incubated with rabbit anti-β-actin (bs-0061R, Bioss, China) as a loading control. After three times of washing every 10 min with PBST, membranes were incubated with goat anti-rabbit IgG (Abcam, UK) for 2 h at room temperature. Protein signal was detected using a 3,3′-diaminobenzidine (DAB) electrochemiluminescence system (Beyotime, Jiangsu, China). Optical density was measured using ImageJ software (NIH, Bethesda, MD, USA).
3
Protein Expression Analysis by Western Blot
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Took out the preserved sample, added RIPA protein lysate and protease inhibitor PMSF, fully lysed, 12,000 × g, centrifuge at 4 °C for 15min. Next, collected the supernatant, extracted the total protein, and used BCA protein detection kit (BioTek) to detect the protein concentration. Then added the collected protein supernatant to 5 × Loading Buffer, fully mixed, denature at 98 °C for 15min and store at -20 °C. The Western blot technique (Xiao et al., 2018 (link)) developed by our laboratory was used to separate the protein by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Atlanta, GA, USA). The electrotransfer solution was washed away with 1× trimethylaminomethane buffer saline (TBST) and was sealed with TBST which contained 5% skimmed milk powder for 1 h. The antibody was incubated with rabbit polyclonal antibody StAR (bs-3570R, 1:500, Bioss) P450scc (bs-10099R, 1:500, Bioss) PGR (bs-23376R, 1:500, Bioss) with β-actin (bs-0061R, 1:3,000, Bioss) as an internal reference, overnight at 4 °C, after washing, goat anti-rabbit secondary antibody (bs-0295G-HRP, Bioss) at a dilution ratio of 1:3,000, at 37 °C for 1 h. Immune complexes were detected with enhanced chemiluminescence solution (Abnova, Taibei, Taiwan), and the expression was quantified using ImageJ (National Institutes of Health).
4
Western Blot Analysis of APP and BACE-1
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The extracted proteins were separated by electrophoresis with the 10% SDS-PAGE. Gel was run at 80V for 20 min and 120V for 60 min until samples run off the gel and then transferred onto PVDF membranes at 4°C at 80V for 1.5 h. The target protein APP and BACE-1 were measured using the primary antibody of anti-APP (Bioworld, BS6418, 1 : 100) and BACE1 (Proteintech 12807-1-AP, 1 : 100) and then incubated at 4°C overnight. After being washed three times with TBST, corresponding secondary antibody was used at a dilution of 1 : 2000 (bs-40295G, bs40296 G, Bioss, China), followed by visualization with ECL kit (mixed with 1 : 1, PE0010, Solarbio, China). The exposure was completed in dark room with chemiluminescence gel imaging system (C600, Azure Biosystems, USA). The antibody against GAPDH (1 : 2000, TA-08, Zsbio, China) and β-actin (1 : 2000, bs-0061R, Bioss, China) were used as internal controls. Quantitative results were expressed as a ratio of APP to β-actin and ratio of BACE-1 to GAPDH and then compared in each group to measure relative changes.
5
SDS-PAGE Analysis of APP, BACE1, and PKA
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The extracted proteins were separated by electrophoresis with 10% SDS-PAGE. Gel was run at 80 V for 20 min and 120 V for 60 min and then transferred onto PVDF membranes at 4°C at 80 V for 1.5 h. The target proteins APP, BACE1, and PKA were measured by incubating with the primary antibodies against APP (1 : 2000, MAB348, Millipore, USA), BACE1 (1 : 2000, ab183612, Abcam, USA), p-PKA (1 : 2000, ab32390, Abcam, USA), and PKA (1 : 2000, ab75993, Abcam, USA) at 4°C overnight. After washing three times with TBST, the corresponding secondary antibody was used at a dilution of 1 : 2000 (bs-40295G, bs40296G, Bioss, China), followed by visualization with the ECL kit (mixed with 1 : 1, PE0010, Solarbio, China). The exposure was completed in a dark room with the chemiluminescence gel imaging system (C600, Azure Biosystems, USA). The antibodies against GAPDH (1 : 2000, TA-08, Zsbio, China) and β-actin (1 : 2000, bs-0061R, Bioss, China) were used as internal controls. Quantitative results were expressed as a ratio of APP to GAPDH, BACE1 to β-actin, and p-PKA to PKA and then compared in each group to measure relative changes.
6
Immunoblotting and Immunohistochemistry Protocols
7
Western Blot Analysis of Protein Biomarkers
8
Western Blot Analysis of Protein Targets
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RIPA buffer (Beyotime) was utilized for extracting total protein from serum and cells. Then, the protein samples were separated by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, United States). After that, the membranes were incubated with anti-CyclinD1 (1:2,000, bs-20596R, Bioss), anti-Cleaved-caspase3 (anti-Cleaved-casp3, 1:2,000, bs-20364R, Bioss), anti-SOX4 (1:1,000, bs-11208R, Bioss), or anti-β-actin (1:20,000, bs-0061R, Bioss) at 4°C overnight. Goat Anti-rabbit IgG antibody (1:2,000, bs-0295G, Bioss) was used to incubate with the membranes for 2 h. After that, protein signal was detected by BeyoECL Plus reagent (Beyotime).
9
Western Blot Analysis of Autophagy Markers
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Lung tissue was lysed in RIPA lysis buffer with protease inhibitor cocktail and centrifuged at 16,000 g for 10 min, and the supernatant was collected. Then, a bicinchoninic acid assay kit
(Thermo Fisher Scientific) was employed to determine protein contents. The protein draws were separated by 6%, 10%, or 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and
then transferred onto polyvinylidene fluoride. The membranes were incubated with the blocking buffer for 1 h at room temperature and then incubated with the primary antibodies. The primary
antibodies used were anti-IRG1 (1:1,000, 17805, CST), anti-SQSTM1/p62 (1:1,000, 5114T, CST), anti-LC3A/B (1:1,000, 12741T, CST), anti-β-actin (1:5,000, bs-0061R, Bioss). The membranes were
incubated with the primary antibodies overnight at 4°C, followed by washing 5 times in phosphate-buffered saline Tween (PBS-T) for 5 min. They were then incubated with a horseradish
peroxidase-conjugated secondary antibody for 1 h and then washed 5 times for 5 min in PBS-T. Finally, Amersham ECL Select Western Blotting Detection Reagent was used for visualization.
(Thermo Fisher Scientific) was employed to determine protein contents. The protein draws were separated by 6%, 10%, or 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and
then transferred onto polyvinylidene fluoride. The membranes were incubated with the blocking buffer for 1 h at room temperature and then incubated with the primary antibodies. The primary
antibodies used were anti-IRG1 (1:1,000, 17805, CST), anti-SQSTM1/p62 (1:1,000, 5114T, CST), anti-LC3A/B (1:1,000, 12741T, CST), anti-β-actin (1:5,000, bs-0061R, Bioss). The membranes were
incubated with the primary antibodies overnight at 4°C, followed by washing 5 times in phosphate-buffered saline Tween (PBS-T) for 5 min. They were then incubated with a horseradish
peroxidase-conjugated secondary antibody for 1 h and then washed 5 times for 5 min in PBS-T. Finally, Amersham ECL Select Western Blotting Detection Reagent was used for visualization.
10
Western Blotting Protocol for HIF-1α and LDHA
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Western blotting was performed as previously described [23 (link)]. Briefly, a polyvinylidene difluoride membrane from Millipore was used to incubate the samples with the primary HIF-1α (rabbit, 1:1000, MA1–516, Thermo Fisher, USA), LDHA (mouse, 1:500, sc137244, Santa Cruz, USA) and β-actin (rabbit, 1:2000, bs-0061R, Bioss, China). The primary Antibodies were labeled with corresponding horseradish peroxidase-conjugated secondary antibodies, and protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL). The EC3 Imaging System (Uplant, UVP) was used to obtain blot images directly from the polyvinylidene difluoride membrane. The Western blot data were quantified using Image-Pro Plus version 6.0.
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