We obtained the tissue microarray from the Outdo Biotech company (HLugA180Su08, Shanghai, China) and the ethics was approved. Anti‐CFL1 antibody (rabbit, 10960‐1‐AP), anti‐PABPC1 antibody (rabbit, 10970‐1‐AP), anti‐CCDC85B antibody (rabbit, 18282‐1‐AP, Proteintech, China), anti‐PFN1 (mouse, 67390‐1‐Ig, Proteintech, China), anti‐HSP90AA1 (rabbit, 13171‐1‐AP, Proteintech, China), anti‐HMGB1 (rabbit, 10829‐1‐AP, Proteintech, China) and anti‐RPS15 (rabbit, 14957‐1‐AP, Proteintech, China) were used as the primary antibody. The secondary antibody (GB23301, GB23303, Servicebio, China) was subsequently used for incubation and tyramide signal amplification (TSA) (FITC‐TSA, CY3‐TSA, 594‐TSA, 647‐TSA, Servicebio, China). The nuclei were stained with 4′,6‐diamidino2‐phenylindole dihydrochloride (DAPI). The Pannoramic Scanner (3D HISTECH, Hungary) was used for image capture. The quantification of the stained markers was performed.
Anti hmgb1
Manufactured by Proteintech
Sourced in China, United States
Anti-HMGB1 is a laboratory reagent used for the detection and quantification of the HMGB1 protein. HMGB1 is a nuclear protein involved in various cellular processes. This product can be used in techniques such as ELISA, Western blotting, and immunohistochemistry to measure HMGB1 levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Pannoramic scanner, by 3DHISTECH (1 mentions)
Anti pfn1, by Proteintech (1 mentions)
Anti hsp90aa1, by Proteintech (1 mentions)
Hluga180su08, by Shanghai Outdo Biotech Co. (1 mentions)
Eclipse ti, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Beyotime (1 mentions)
Anti transferrin, by Abcam (1 mentions)
Anti pi3 kinase p110γ, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Proteintech (1 mentions)
Anti tlr4, by Santa Cruz Biotechnology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
9 protocols using anti hmgb1
1
Quantitative Multiplex Immunofluorescence Tissue Proteomics
2
Neutrophil and Endothelial Cell Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GSDMD and HMGB1 expression in neutrophils and ICAM-1 and IL-8 expression in HUVECs was assessed via immunofluorescent staining. In brief, cells were fixed for 20 min with 4% paraformaldehyde and permeabilized for 30 min with 0.2% Triton X-100 at room temperature, then blocked with 50% goat serum (Beyotime Institute of Biotechnology) in PBS for 30 min at 37°C and incubated overnight with anti-GSDMD (cat. no. 20770-1-AP; 1:100 dilution; Proteintech Group, Inc.), anti-HMGB1 (cat. no. bs-55098R; 1:50 dilution; Bioss) or anti-ICAM-1 (cat. no. WL02268) or anti-IL-8 (cat. no. WL03074; both 1:50 dilution; both from Wanleibio Co., Ltd.) at 4°C. Cells were then stained with an AF488-conjugated secondary antibody (cat. no. R37118; 1:1,000 dilution; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h and DAPI (1 μg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) was applied for nuclear staining for 10 min at room temperature. Cells were rinsed with PBS and imaged via a fluorescence microscope (ECLIPSE Ti; Nikon Corporation).
3
Quantitative Protein Analysis of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins of cells and tissues were extracted and measured by Western blot assay using primary antibodies of anti-Slug, anti-E-Cadherin, anti-p-Akt (Thr308), anti-Akt (pan), anti-PI3-kinase p110γ (Cell Signaling Technology, 1:1000), anti-TLR4 (Santa Cruz; 1:1000), anti-HMGB1, anti-TNF-α (Proteintech Group, Inc., Rosemont, IL, USA; 1:1000), anti-transferrin (Abcam, 1:1000) and anti-β-actin (Beyotime; 1:1000). Proteins transferred on a PVDF membrane were incubated with primary antibodies overnight at 4°C and labeled with secondary HRP-conjugated antibodies (Beyotime; 1:1000) for 2 h at room temperature. The protein bands were observed using a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using the Quantity One software (Bio-Rad Laboratories).
4
Immunocytochemistry of α-SMA and HMGB1 in 16HBE cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 16HBE cell line (American Type Culture Collection) was cultured with RPMI-1640 containing 10% FBS (both from Gibco; Thermo Fisher Scientific, Inc.) in a cell-culture incubator at 37°C with 5% CO2. At 90% confluence, cells were placed on a coverslip in six-well plates at a density of 2×105 cells/well. MK2206 (1 µg/ml) or DEX (1 µg/ml) was added to the medium 1 h before TDI (60 µg/ml) exposure for 6 h. For immunocytochemistry, cells were fixed in 4% formaldehyde-PBS at room temperature for 10 min, permeabilized in 0.5% Triton X-100 at room temperature for 5 min and blocked with 5% (w/v) BSA for 1 h at room temperature, followed by overnight incubation with anti-α-SMA (1:100; cat. no. 23081-1-AP; ProteinTech Group, Inc.) and anti-HMGB1 (1:100; cat. no. 66525-1-Ig; ProteinTech Group, Inc.) antibodies at 4°C. The next day, cells were washed in three times PBS and incubated with Alexa Fluor® 488-labeled goat anti-mouse IgG antibody (1:1,000; cat. no. SA00004-11; ProteinTech Group, Inc.) at room temperature for 1 h in the dark. The nuclei were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature for 10 min. The immunofluorescence of α-SMA and HMGB1 was observed at ×200 magnification using a laser-scanning confocal microscope (Olympus Corporation). The immunofluorescence was quantified by ImageJ software (1.48v; National Institutes of Health).
5
Exosomal Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and exosomes were lysed and collected in a protein lysate, and the concentration was measured using a BCA kit (Thermo Fisher Scientific, 23327). Protein samples (30 μg) were electrophoresed, transferred to a nylon membrane and blocked with a blocking buffer. Finally, the cells were incubated with the primary antibody at 4 °C overnight. The antibodies used were anti-HMGB3 (cat:D160490, Sangon Biotech, China), anti-flotillin-1 (cat:ab41927, Abcam, USA), anti-actinin-4 (cat:ab108198, Abcam, USA), anti-Alix (cat:ab186429, Abcam, USA), anti-CD9 (cat:ab92726, Abcam, USA), anti-GAPDH (cat:10494-1-AP, Proteintech, China), anti-E-cadherin (cat:A3044, ABclonal, China), anti-Vimentin (cat:10366-1-AP, Proteintech, China), anti-N-cadherin (cat:A3045, ABclonal, China), anti-HMGB1 (cat:10829-1-AP, Proteintech, China), anti-HMGB2 (cat:14597-1-AP, Proteintech, China) and anti-HMGB4(cat:12787-1-AP, Proteintech, China). Following incubation with a goat anti-rabbit secondary antibody, the immunoreactive proteins were detected with ECL western blotting detection reagents (Millipore, WBKLS0500).
6
Protein Expression Analysis in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein in tissues and cell lines was extracted in RIPA reagent (Beyotime, Shanghai, China) supplemented with protease inhibitor PMSF (Sigma‐Aldrich). Then, an aliquot of protein sample (20 μg) was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF; Millipore) membrane. After blocking in 5% skin milk, the membranes were incubated in primary antibodies from Proteintech (Deansgate, Manchester, UK) including anti‐HMGB1 (10829‐1‐AP, 1: 2000), antiproliferating cell nuclear antigen (PCNA; 10 205‐2‐AP, 1: 10000), anti‐E‐cadherin (E‐cad; 20 874‐1‐AP, 1: 50000), anti‐N‐cadherin (N‐cad; 22 018‐1‐AP, 1: 10000), and anti‐GAPDH (10494‐1‐AP, 1: 40000) at 4°C for overnight, and in secondary antibody anti‐rabbit IgG‐HRP (sc‐2357, 1: 5000) from Santa Cruz (Shanghai, China) at room temperature for 2 hours. The protein blots were developed using ECL reagent (Millipore), and captured by Chemiluminescence (Thermo Fisher Scientific). GAPDH was the internal control.
7
Western Blot Analysis of Cerebral Ischemia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were prepared from the same regions of ischemic cerebral hemisphere (bregma 0 to + 2 mm) from different treatment groups, or serum proteins after magnetic separation. Fifty micrograms of total proteins were separated on 8 to 12% SDS-PAGE gel. After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies including anti-occludin (1:1000; Invitrogen, Camarillo, CA), anti-COX-2 (1:1000; CST, Beverly, MA), anti-IL-1β, anti-iNOS (1:200; Santa Cruz Biotech, CA), anti-β-actin (1:500; ZSGB-Bio, Beijing, China) or anti-HMGB1 (1:1000; Proteintech, UK), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotech). Bands were detected by ECL advance Western blotting detection reagents (Millipore, Billercia, MA). The band intensities were normalized to β-actin using the ImageJ.
8
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from lung tissues and cells. BCA protein concentration kit (Beyotime) was used to detect protein concentration. Equal amount (20 µg, 20 μl per lane, 1 μg/μl) of protein was separately by 10% SDS-PAGE. After transferred onto polyvinylidene difluoride membranes, the proteins were incubated with primary antibodies at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated anti-rabbit/mouse secondary antibodies (1:5000, Beyotime) for 2 h at room temperature. Then, the membranes were visualized with chemiluminescence reagent and the optical density values were analyzed by Gel-Pro-Analyzer software. β-actin was used as an internal control to standardize the target protein levels. The primary antibodies used in the present study were: anti-HMGB1 (1:1000, Proteintech, Wuhan, China), anti-TLR4 (1:1000, Proteintech), anti-TLR5 (1:1000, Proteintech), anti-Myd88 (1:1000, Affinity, Cincinnati, OH, USA), anti-P65/P-P65 (Ser 536) (1:1000, Affinity), anti-IκBα/P-IκBα (Ser 32/Ser 36) (1:1000, Affinity), and anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
9
Quantification and Analysis of Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured GC cells were collected and washed three times with PBS, and then, RIPA buffer containing protease inhibitor (Thermo Scientific, USA) was added to the cells according to the number of cells. The cell lysates were dissolved on ice for 30 minutes and then ultrasonically crushed and quantified by using BCA assays. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Next, the membranes were blocked with 5% nonfat milk for 2 h, and the primary antibodies were incubated on a shaker in a refrigerator at 4°C overnight. Then, the membranes were washed with TBST, and HRP-conjugated secondary antibodies were added for 1-1.5 h at room temperature. The target proteins were detected with a Bio-Rad ChemiDoc XRS System. Densitometric analysis was performed by using Bio-Rad Image Lab software. The primary antibodies and dilution multiples involved in this study were as follows: anti-FBXW7 (1 : 1000, Abcam, USA), anti-BGN (1 : 1000, CST, USA), anti-HMGB1 (1 : 1000, Proteintech, USA), anti-H3 (1 : 1000, CST, USA), and anti-GAPDH (1 : 5000, Santa Cruz, CA). The secondary antibodies were diluted at 1 : 5000.
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!