HepG2 cells were treated with Hcy (0–100 µM) for 24 h at 37°C. Cells were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology) containing leupeptin. Protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Cellular protein samples (30 µg) were separated by 10% SDS-PAGE as previously described (19 (link)). Proteins were transferred onto nitrocellulose membranes and the membranes were incubated with an anti-Nrf2 polyclonal antibody (1:500; bs-1074R, BIOSS, Beijing, China) or an anti-β-actin monoclonal antibody (1:3,000; TA-09; Zhongshan Golden Bridge Biotechnology, Beijing, China) overnight at 4°C, followed by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1: 5,000; ZB-2301, Zhongshan Golden Bridge Biotechnology). Protein bands were visualized by enhanced chemiluminescence (HaiGene Bio Inc.). Densitometric analysis of band intensity was calculated using Quantity One software, version v4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Bs 1074r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-1074R is a laboratory device designed for the detection and quantification of biomolecules. It utilizes a robust and reliable detection method to provide accurate and consistent results. The core function of the Bs-1074R is to facilitate the analysis of various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ta 09, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Quantity one software version v4, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Zb 2301, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Ab183781, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Biotinylated goat anti rabbit igg, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Ba2305, by Boster Bio (1 mentions)
Bs 0549r, by Bioss Antibodies (1 mentions)
Bs 0578r, by Bioss Antibodies (1 mentions)
Imagej software, by Merck Group (1 mentions)
A12477 2, by Boster Bio (1 mentions)
Nucleoprotein extraction kit, by Sangon (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Bs 21501r, by Bioss Antibodies (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Bs 20412r, by Bioss Antibodies (1 mentions)
9 protocols using bs 1074r
1
Hcy-Induced Nrf2 Expression in HepG2 Cells
2
Histological Analysis of Nrf2, FTH1, and GPX4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Once sacrificed, rats underwent decapitation, the cerebrum was extracted and tissue from peri-haemorrhagic areas was harvested from the various cohorts. Following embedding in paraffin, the specimens were sliced into 5-µm sections; xylene was utilised for dewaxing. The sections were then soaked in antigen repair solution and a low flame was applied for 10 min. After cooling to the ambient temperature, the samples underwent incubation in 3% hydrogen peroxide; goat serum was used for blocking. Threefold 5-min washing in phosphate-buffered saline was performed at each stage of the process.
The sections then underwent overnight incubation at a temperature of 4 °C with primary antibodies, i.e. rabbit anti-Nrf2 (1:200, no. bs-1074R, Bioss), rabbit anti-FTH1 (1:400, no. ab183781, Abcam) and rabbit anti-GPX4 (1:300, no. ab125066, Abcam). Incubation for 30 min at 37 °C with the equivalent secondary antibody, i.e. biotinylated goat anti-rabbit IgG (1:200, no. A0277, Beyotime) was performed. Lastly, labelling with horseradish, produced in DAB, was undertaken. The samples were soaked in distilled water and then haematoxylin counterstaining was conducted, followed by desiccation and sealing of the sections. A microscope (DP73, Olympus Corporation, Tokyo, Japan) was employed to acquire images at × 400 magnification.
The sections then underwent overnight incubation at a temperature of 4 °C with primary antibodies, i.e. rabbit anti-Nrf2 (1:200, no. bs-1074R, Bioss), rabbit anti-FTH1 (1:400, no. ab183781, Abcam) and rabbit anti-GPX4 (1:300, no. ab125066, Abcam). Incubation for 30 min at 37 °C with the equivalent secondary antibody, i.e. biotinylated goat anti-rabbit IgG (1:200, no. A0277, Beyotime) was performed. Lastly, labelling with horseradish, produced in DAB, was undertaken. The samples were soaked in distilled water and then haematoxylin counterstaining was conducted, followed by desiccation and sealing of the sections. A microscope (DP73, Olympus Corporation, Tokyo, Japan) was employed to acquire images at × 400 magnification.
3
Immunohistochemical Analysis of Nrf2, Fibronectin, and C5b-9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, following polyclonal or monoclonal Abs (pAbs or mAbs) were used for immunohistochemical analyses in the present study: rabbit anti-human Nrf2 pAbs (bs-1074R, Bioss antibodies, Woburn, MA), rabbit anti-human fibronectin pAbs (15613-1-AP, Proteintec, Rosemont, IL), rabbit anti-human C5b-9 pAbs (bs-2673R, Bioss antibodies).
4
Western Blot Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer (catalogue number: P0013C, Beyotime, Haimen, China) was adopted for total protein extraction from mouse hearts or HL‐1 cells. For isolation of nuclear protein, the Nucleoprotein Extraction Kit (catalogue number: C500009, Sangon Biotech, Shanghai, China) was used. After protein concentration quantification, protein samples with equal amount (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skim milk for 1 h at room temperature. The membranes were probed with primary antibodies against ATF4 (bs‐1531R, 1:500, Bioss, Beijing, China), collagen I (bs‐0578R, 1:500, Bioss), collagen III (bs‐0549R, 1:500, Bioss), HIPK2 (bs‐6353R, 1:500, Bioss), Nrf2 (bs‐1074R,1:100, Bioss), HO‐1 (BM4010, 1:500, BOSTER, Wuhan, China), Smurf2 (#12024, 1:1000, Cell Signaling Technology, Trask Lane Danvers, USA), histone H3 (A12477‐2, 1:500, BOSTER), and β‐actin (BA2305, 1:2000, BOSTER) at 4°C overnight. Subsequently, the secondary antibody (#7074, 1:3000, Cell Signaling Technology) was applied for 1 h at room temperature. The immune blots were developed using the enhanced chemiluminescence (Millipore, USA) and subjected to densitometry analysis using ImageJ software (Bethesda, USA). Three independent experiments were conducted.
5
Protein Isolation and Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated from tissues and H9c2 cardiomyocytes at 72 h after transfection with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime; China) supplemented with phenylmethanesulfonyl fluoride (Beyotime; China). The protein concentration was determined with a Bicinchoninic Acid (BCA) Protein Assay Kit (KeyGen; China). Equal protein samples (3.75 μg) were separated by 10% twelve alkyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature. The blots were incubated with primary antibodies against TBX1 (1 : 1000, bs-21501R, Bioss), TGF-β2 (1 : 1000, bs-20412R, Bioss), NRF2 (1 : 500, bs-1074R, Bioss), GPX4 (1 : 1000, DF6701, Affinity), HO-1 (1 : 5000, sc-390991, Santa Cruz), NOX4 (1 : 1000, 14347-1-AP, Proteintech), ACSL4 (1 : 1000, AP2536B, Abcepta), and GAPDH (1 : 10000, AF7021, Affinity) overnight and then with a goat anti-rabbit secondary antibody (1 : 10000, S0001, Affinity) for 1 h at room temperature. Electrochemiluminescence (ECL) Western Blotting Substrate (Tanon; China) was used to visualize specific proteins, and the blots were analysed using a ChemiDoc Touch Imaging System (Bio-Rad, USA). ImageJ software was used to measure the protein band intensity, and GAPDH was used as the internal reference.
6
Immunohistochemical Analysis of Key Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the sections were deparaffinized and rehydrated, the experiments were performed following the IHC kit instructions (kit-9720, MXB, China) for MMP13 (18165-1-AP; 1:200; Proteintech), Nrf2 (bs-1074R; 1:500; Bioss), P53 (A0263; 1:100; Abclonal), SLC7A11 (DF12509; 1:100; Affinity Biosciences), Col2a1 (28459-1-AP; 1:1000; Proteintech), p-NF-κB p65 (bs-5662R; 1:200; Bioss), and GPX4 (67763-1-Ig; 1:2000; Proteintech). A series of primary antibodies were added to the sections of the four groups, and then all sections were incubated in a wet box at 4 °C overnight. The following day, the sections were incubated at room temperature for 1 h with a secondary antibody, and a DAB solution (dab-0031, MXB, China) was added for visualization. Finally, after neutral resin blocking, they were photographed under a microscope. Five fields were randomly selected for each sample, and the expression of each index was examined and averaged for statistical analysis.
7
Western Blot Analysis of MUC13 in Transfected GC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from transfected GC cells using RIPA buffer (Thermo Fisher Scientific). A BCA protein quantification kit (Beyotime, China) was used to determine the protein concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for protein separation. The gels were transferred to poly (polyvinylidene fluoride) (PVDF) membranes and blocked with 5% nonfat milk for 1 h at room temperature. These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase‑conjugated goat anti‑rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C. Finally, the protein bands were visualized using enhanced chemiluminescence (Pierce, Thermo Fisher Scientific, USA). The grayscale of the protein bands was analyzed using ImageJ software.
8
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were lysed with RIPA buffer, separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The proteins were then immunoblotted with the primary antibody: anti-SGK1 (bms-33275 M, Bioss), anti-pSTAT3 (bs-16558R, Bioss), anti-STAT3 (ab109085, Abcam), anti-NRF2 (bs-1074R, Bioss), anti-ERK1 + ERK2 (ab184699, Abcam), anti-pERK1 + pERK2 (ab214036, Abcam), anti-β-actin (4970, Cell Signaling Technology), anti-MPO (ab208670, Abcam), anti-CitH3 (ab281584, Abcam), anti-NE (bs-6982R, Bioss), anti-PAD4 (ab214810, Bioss). This was followed by color development using horseradish peroxidase (HRP)-enhanced chemiluminescence (ECL) assays.
9
Protein Expression Analysis of BMECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of BMECs was extracted by using RIPA buffer (Beyotime, Shanghai, China). The protein samples were quantified via a BCA assay kit (Beyotime, Shanghai, China). Subsequently, 30 μg protein in each group was separated by using SDS-PAGE and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked in 5% skim milk. Next, the membranes were incubated overnight with primary antibody against ZO-1 (1 : 1000, ab221547, Abcam, Cambridge, MA, USA), Occludin (1 : 1000, ab216327, Abcam), Claudin 5 (1 : 1000, ab131259, Abcam), Nrf2 (1 : 1000, bs-1074R, Bioss, Beijing, China), and HO-1 (1 : 1000, ab52947, Abcam) at 4°C and then interacted with secondary antibody for 2 hours. Protein bands were revealed by the enhanced chemiluminescence (ECL) method. β-Actin (1 : 5000, Abcam, ab227387) was used as a loading reference.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!