Total protein was extracted by RIPA buffer (Solarbio, Beijing, China) containing protease inhibitors (PMSF) (Solarbio, Beijing, China), while nuclear protein was extracted using a Nuclear Protein Extraction kit (Solarbio, Beijing, China). The protein concentrations were determined using a BCA Protein Assay kit (Solarbio, Beijing, China). The tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Pall Co, USA) at a voltage of 90V. Blotted membranes were blocked with 5% BSA for 1.5 h at room temperature and incubated overnight at 4°C with anti-FAS(#3180, Cell Signaling Technology), anti-SREBP-1(#NB100-2215, Novus), anti-NRF2(#12721, Cell Signaling Technology), anti-AHR (#sc-13308, Santa Cruz Biotechnology), anti-Lamin-B2(#12255, Cell Signaling Technology) and anti-β-Actin(#3700, Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) for 1 h at room temperature. Fluorescent bands were visualized and photographed using an SYNGENE CGQ/D2 GEL-Image System (Bio-Rad, America).
Anti ahr
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-AHR is a laboratory reagent used for detecting and quantifying the expression of the aryl hydrocarbon receptor (AhR) protein. AhR is a transcription factor that plays a crucial role in cellular responses to various environmental contaminants and signaling pathways. Anti-AHR can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to analyze AhR levels and distribution in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti fas, by Cell Signaling Technology (1 mentions)
Nuclear protein extraction kit, by Solarbio (1 mentions)
Anti srebp 1, by Novus Biologicals (1 mentions)
Anti lamin b2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti h3k4me3, by Cell Signaling Technology (1 mentions)
Anti α smooth muscle actin, by Santa Cruz Biotechnology (1 mentions)
Col1a1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti kdm5b, by Cell Signaling Technology (1 mentions)
Anti h3k27ac, by Cell Signaling Technology (1 mentions)
Bioruptor twin, by Diagenode (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Power sybr green mix, by Thermo Fisher Scientific (1 mentions)
Anti taz, by Cell Signaling Technology (1 mentions)
Mouse igg, by Santa Cruz Biotechnology (1 mentions)
7 protocols using anti ahr
1
Protein Extraction and Western Blot Analysis
2
Antibody-based Protein Expression Analysis
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Anti‐KDM5B, Ani‐KDM5C, and Anti‐H3K4me3 antibodies were from Cell Signaling Technology (Beverly, MA). Anti‐β‐actin, anti‐AhR, anti–α‐smooth muscle actin and anti–collagen type I alpha 1 chain (Col1a1) antibodies were from Santa Cruz.
3
ChIP-seq Protocol for Histone Modifications
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Five million H1975 cells were seeded in 15 cm dishes and transfected with the siRNAs (siNT and siMESH1s) for 72 h before cross-link and collection of the cell lysates. Myers Lab ChIP-seq Protocol v011014-Adherent cells was then followed (https://www.encodeproject.org/documents/6ecd8240-a351-479b-9de6-f09ca3702ac3/@@download/attachment/ChIP-seq_Protocol_v011014.pdf) . Sonication condition was optimized to the High mode, 30 s on/30 s off at 4 °C for 45 min using the Bioruptor Twin (Diagenode) sonicator. Dynabeads Protein G (Invitrogen, #10003D) and anti-H3K27Ac (Cell Signaling Technology, #8173), anti-TAZ (Cell Signaling Technology, #70148), anti-AHR (Santa Cruz Biotechnology, #sc-133088 X) or rabbit IgG (negative control: Santa Cruz Technology, sc-66931), mouse IgG (Santa Cruz Technology, sc-2025) were used for pull-down in this study as instructed. Pulled down DNA was then mixed with primers targeting the TAZ promoter, or YAP promoter, or TAZ-proximal heterochromatin region together with the Power SYBRGreen Mix (ThermoFisher Scientific, #4367659) to undergo the qPCR reaction as described above. Primers were designed following the guidelines on the Michigan University Nutritional Sciences website (http://bridgeslab.sph.umich.edu/protocols/index.php/RT-PCR_primer_design_for_ChIP ). n = 3 biologically independent replicates.
4
Signaling Pathway Analysis in Cell Culture
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Culture media and chemical compounds were purchased from Life Technologies (Grand Island, NY, USA). Antibodies against phosphor (P)‐EGFR (1:1,000), EGFR (1:1,000), pSTAT3 (1:1,000), STAT3 (1:1,000), pAKT (1:1,000), pERK (1:1,000), IL‐18 (1:200), vimentin (1:1,000), and Ki‐67 (1:1,000) were purchased from Cell Signaling (Temecula, CA, USA). Anti‐AKT (1:1,000), anti‐ERK (1:1,000), anti‐TMPRSS2 (1:500), anti‐AhR (1:500), and anti‐Lamin B (1:1,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against AhR (1:500) was purchased from GeneTex (Irvine, CA, USA). The antibody against β‐actin (1:1,000) and HA (1:1,000) was purchased from Sigma (St. Louis, MO, USA). Alexa Fluor® 488 goat anti‐mouse IgG (1:1,000) was purchased from Life Technologies (Waltham, MA, USA). CH223191 was purchased from Sigma (St. Louis, MO, USA).
5
UVB-induced Antioxidant Response in HaCaT Cells
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HaCaT cells were seeded in 10-cm dishes (1 × 106 cells/dish) and incubated at 37°C for 24 hours in a 5% CO2 incubator. The cells were then starved with serum-free medium for 24 hours, before the medium was changed with 1,8-cineole for 1 hr. The cells were then exposed to UVB irradiation (0.04 J/cm2) and incubated for 30 min. The HaCaT cells were lysed in M-PER lysis buffer (Pierce, Rockford, IL, USA) containing protease and phosphatase inhibitors. The cell lysate containing 500 mg of protein was incubated with anti-IgG (Cell Signaling Biotechnology; Beverly, MA, USA) or anti-AhR (Santa Cruz, CA, USA) overnight at 4°C. Then, 20 μL of a 50% slurry of protein G agarose beads (Pierce, Rockford, IL, USA) was added to the samples followed by incubation for 2 hours at 4°C. The samples were centrifuged for 1 min at 14,000 g and washed with lysis buffer five times. The pellets were resuspended with 20 uL of 3x SDS sample buffer and the samples were heated at 95°C for 5 min for subsequent Western blotting assay.
6
Immunohistochemical Analysis of AHR, LRIG1, and EGFR
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The lung specimens of wild-type and AHR-KO mice were a gift from Dr. Lee CC [54 (link)]. The human lung disease and normal tissue array were obtained from US Biomax, Inc., Derwood, MD, USA (catalog no. LUD481). The expression patterns of AHR, LRIG1, and EGFR were detected by using specific primary antibodies (anti-AHR, Santa Cruz, catalog no. sc-74571; anti-LRIG1 and anti-EGFR, Genetex, catalog no. GTX119485 and GTX121919, respectively) according to the standard protocol of Bio-Check Laboratories Ltd. (New Taipei City, Taiwan). Peroxidase-labeled specimens were observed on a Nikon light microscope equipped with a Polychrome-III camera (YC technology, New Taipei City, Taiwan) and Image Eye software (FMJ Software, Stockholm, Sweden).
7
Osteoblast Differentiation and Signaling
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All culture media components, including α-MEM, fetal bovine serum, and antibiotic–antimycotic (100×), were acquired from Gibco (Las Vegas, NV, USA). An osteoblast differentiation medium containing L-ascorbic acid, β-glycerophosphatase, and dexamethasone was obtained from Sigma (St. Louis, MO, USA). The indoxyl sulfate (IS) and resveratrol (RSV) used in the osteoblast treatment were acquired from Sigma (St. Louis, MO, USA). The primary antibodies were obtained as indicated: anti-AhR (Santa Cruz, Dallas, TX, USA), anti-CYP1A1 (GeneTex, Hsinchu City, Taiwan), anti-CYP1B1 (GeneTex, Hsinchu City, Taiwan), anti-MAPK series (ERK1/2, p38, JNK, phospho-ERK1/2, phospho-p38, and phospho-JNK) (Cell Signaling, MA, USA), anti-Runx2 (Abcam, Cambridge, UK), anti-collagen 1 (GeneTex, Hsinchu City, Taiwan), anti-osteocalcin (Santa Cruz, Dallas, TX, USA), and anti-β Actin (Proteintech, Rosemont, IL, USA). Horse Radish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G and anti-mouse immunoglobulin G were obtained from the Proteintech Group (Chicago, IL, USA). The qPCR reagents included the RNA Extractor kit (Tools, Taiwan), DNase I Amplification Kit (Invitrogen, USA), iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and Simply Green qPCR Master Mix (GeneDireX Inc., Taichung City, Taiwan).
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