Log In Sign up
The largest database of trusted experimental protocols

Adar1

Manufactured by Santa Cruz Biotechnology
Visit Supplier
Sourced in United States

ADAR1 is a protein that catalyzes the deamination of adenosine to inosine within double-stranded RNA molecules. This enzymatic activity is essential for the regulation of gene expression and the modulation of RNA processing.

Automatically generated - may contain errors

Lab products found in correlation

Gapdh, by Fortis Life Sciences (3 mentions) Pkr thr 446 p, by Abcam (3 mentions) Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions) Image lab software, by Bio-Rad (3 mentions) Chemidoc imaging system, by Bio-Rad (3 mentions) Clarity western ecl substrate, by Bio-Rad (3 mentions) Tgx acrylamide stain free gels, by Bio-Rad (3 mentions) Stat2, by Santa Cruz Biotechnology (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Il 1β, by Santa Cruz Biotechnology (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Total pkr, by Abcam (1 mentions) Caspase 1, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Merck Group (1 mentions) Nlrp3, by Adipogen (1 mentions) Bcl2 associated x (bax), by Abcam (1 mentions) Phosphatase inhibitor cocktail, by Merck Group (1 mentions) Mettl3, by Abcam (1 mentions) Anti rabbit and anti mouse peroxidase conjugated secondary antibodies, by Santa Cruz Biotechnology (1 mentions)

12 protocols using adar1

1

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins from cell-free supernatants were extracted by methanol/chloroform precipitation as described previously42 (link). Cell extracts were also prepared. Samples were separated on SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin for 2 h and then incubated overnight at 4 °C with antibodies specific for phosphor-Thr466 on PKR (Abcam), total PKR (Abcam), caspase-3 (Cell Signaling), BAX (Abcam), IL-1β (Santa Cruz), caspase-1 (Santa Cruz), NLRP3 (Adipogen), STAT-2 (Santa Cruz), ADAR1 (Santa Cruz) and GAPDH (Sigma) followed by incubation with HRP-conjugated secondary antibody. The quantitative relative expression of proteins were calculated by normalizing the densitometric analysis to GAPDH.
Wang Y., Men M., Xie B., Shan J., Wang C., Liu J., Zheng H., Yang W., Xue S, & Guo C. (2016). Inhibition of PKR protects against H2O2-induced injury on neonatal cardiac myocytes by attenuating apoptosis and inflammation. Scientific Reports, 6, 38753.
+ Open protocol
+ Expand
2

Protein Extraction and Immunoblotting Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total protein extracts were isolated with RIPA lysis buffer in the presence of a protease inhibitor mixture and phosphatase inhibitor cocktail (SIGMA). Protein extracts were quantified with the BCA Protein Assay Kit (Pierce). Equal amounts of total cellular lysates (30 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, analyzed by immunoblotting with the appropriate antibodies and then revealed by ECL (GE Healthcare). The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).
Tassinari V., Cesarini V., Tomaselli S., Ianniello Z., Silvestris D.A., Ginistrelli L.C., Martini M., De Angelis B., De Luca G., Vitiani L.R., Fatica A., Locatelli F, & Gallo A. (2021). ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism. Genome Biology, 22, 51.
+ Open protocol
+ Expand
3

Western Blot Quantification of ADAR1, PKR, and pPKR

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell pellets were lysed and sonicated in RIPA Buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS and 0.5% sodium deoxycholate) with 1× HALT Protease Inhibitor (Pierce). Forty micrograms of protein lysate were resolved on 4%–12% TGX Acrylamide Stain-Free gels (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membrane (Millipore). The membrane was cut into strips corresponding to the molecular weight of proteins of interest. The blots were blocked and then probed with the appropriate primary antibodies: ADAR1 (Santa Cruz Biotechnology, sc-73408), PKR (Cell Signaling Technology, #3072), PKR Thr-446-P (Abcam, ab32036), GAPDH (Bethyl Laboratories, A300–641A). Primary antibodies were detected with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch) and detection was carried out with Clarity Western ECL Substrate (Bio-Rad). Chemiluminescence was imaged using a ChemiDoc imaging system (Bio-Rad). Quantification of immunoblots was performed using Image Lab software (Bio-Rad). The abundance of each protein was normalized to GAPDH abundance. For PKR and pPKR, two separate gels were resolved, transferred, and probed for either PKR or pPKR in addition to GAPDH for both. PKR and pPKR abundance were normalized to GAPDH prior to normalizing pPKR to PKR. Uncropped immunoblot images are available in Supplementary Figs. S1–S5.
Cottrell K.A., Soto-Torres L., Dizon M.G, & Weber J.D. (2021). 8-Azaadenosine and 8-Chloroadenosine are not Selective Inhibitors of ADAR. Cancer Research Communications, 1(2), 56-64.
+ Open protocol
+ Expand
4

Immunoblot Quantification of ADAR1, PKR, and pPKR

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell pellets were lysed and sonicated in RIPA Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate and 0.5% sodium deoxycholate) with 1x HALT Protease Inhibitor (Pierce). Forty micrograms of protein lysate were resolved on 4–12% TGX Acrylamide Stain-Free gels (Bio-Rad). Proteins were transferred to PVDF membrane (Millipore). The membrane was cut into strips corresponding to the molecular weight of proteins of interest. The blots were blocked and then probed with the appropriate primary antibodies: Primary antibodies: ADAR1 (Santa Cruz, sc-73408), PKR (Cell Signaling, #3072), PKR Thr-446-P (Abcam, ab32036), GAPDH (Bethyl, A300–641A). Primary antibodies were detected with horseradish-peroxidase conjugated secondary antibodies (Jackson ImmunoResearch) and detection was carried out with Clarity Western ECL Substrate (Bio-Rad). Chemiluminescence was imaged using a ChemiDoc imaging system (Bio-Rad). Quantification of immunoblots was performed using Image Lab software (Bio-Rad). The abundance of each protein was normalized to GAPDH abundance. For PKR and pPKR, two separate gels were resolved, transferred, and probed for either PKR or pPKR in addition to GAPDH for both. PKR and pPKR abundance were normalized to GAPDH prior to normalizing pPKR to PKR. Uncropped immunoblot images are available in Supplemental Figures 1–5.
Cottrell K.A., Soto-Torres L., Dizon M.G, & Weber J.D. (2021). 8-Azaadenosine and 8-Chloroadenosine are not Selective Inhibitors of ADAR. Cancer Research Communications, 1(2), 56-64.
+ Open protocol
+ Expand
5

Western Blotting for Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Whole-cell lysates for Western blotting were extracted with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease, and phosphatase inhibitor cocktail). Protein concentrations were determined using bicinchoninic acid (Pierce) assays according to the manufacturer's instructions. Cell lysates (30 µg) were loaded onto 8% or 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane, and protein expression was depicted with an enhanced chemiluminescence Western blot detection kit (Amersham Biosciences). The following antibodies were used: LIFR (1:500, Santa Cruz Biotechnology, sc-659), ZEB1 (1:500, Novus Biologicals, NBP1-05987), and ERK2 (1:2000, Santa Cruz Biotechnology, sc-154), ADAR1 (1:1000, Santa Cruz Biotechnology, sc-271854), ADAR2 (1:1000, Genetex, GTX114237), and GAPDH (1:3000, Santa Cruz Biotechnology, sc-25778).
Wang Y., Xu X., Yu S., Jeong K.J., Zhou Z., Han L., Tsang Y.H., Li J., Chen H., Mangala L.S., Yuan Y., Eterovic A.K., Lu Y., Sood A.K., Scott K.L., Mills G.B, & Liang H. (2017). Systematic characterization of A-to-I RNA editing hotspots in microRNAs across human cancers. Genome Research, 27(7), 1112-1125.
+ Open protocol
+ Expand
6

Western Blot Analysis of Cardiac Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cardiac tissues and transfected cells were homogenized in RIPA buffer (Heart, Beijing, China). BCA Protein Assay Kit was used to determine the protein concentrations. Then, 50 μg of total protein per sample was subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a PVDF membrane (BioRad, Hercules, CA, USA). The membrane was then incubated with the primary antibody of interest, washed with PBS-T, and incubated with an appropriate secondary antibody. Protein bands were quantified with ImageQuant software. The β-actin level was used as an internal control. The following primary antibodies were used: ADAR1 (Santa Cruz Biotechnology, Dallas, TX, USA), Dicer (Biorbyt, Cambridge, UK), PTEN (Abcam, Cambridge, UK), BAX (Cell Signaling Technology, Danvers, MA, USA); Bcl-2 (Bioworld, Nanjing, China); β-actin (Cmctag, Shanghai, China); GAPDH (Cmctag, Shanghai, China). Three independent experiments were performed for each sample.
Zhang X., Gao X., Hu J., Xie Y., Zuo Y., Xu H, & Zhu S. (2019). ADAR1p150 Forms a Complex with Dicer to Promote miRNA-222 Activity and Regulate PTEN Expression in CVB3-Induced Viral Myocarditis. International Journal of Molecular Sciences, 20(2), 407.
+ Open protocol
+ Expand
7

Comprehensive Immunoblotting Antibody Panel

Check if the same lab product or an alternative is used in the 5 most similar protocols
ADAR1(Santa Cruz Biotechnology, sc-73408), alpha-Tubulin (Santa Cruz Biotechnology, sc-32293), COIL (Cell Signaling Technology, D2L3J, #14168), DHX9 (Abcam, ab183731), FLAG-M2 (Sigma, F3165), GFP (Chromotek, PAGB1), Myc-tag (Cell Signaling Technology, 9B11, #2276), Phospho-Rpb1 CTD (Ser2) (Cell Signaling Technology, E1Z3G, #13499), PNN (Abcam, ab244250), RBM25 (Sigma, HPA070713-100UL), SC-35 (Sigma, S4045), SRSF1 (SF2/ASF, Santa Cruz Biotechnology, sc-33652), SON (polyclonal rabbit, Sigma, HPA023535), SRRM1 (Abcam, ab221061), SRRM2 (Thermo Fisher Scientific, PA5-66827), SRSF2 (Abcam, ab28428), SRSF7 (MBL, RN079PW), U1-70K (Santa Cruz Biotechnology, sc-390899), U2AF65 (Santa Cruz Biotechnology, sc-53942).
Ilik İ.A., Malszycki M., Lübke A.K., Schade C., Meierhofer D, & Aktaş T. (2020). SON and SRRM2 are essential for nuclear speckle formation. eLife, 9, e60579.
+ Open protocol
+ Expand
8

Immunohistochemical Staining of ADAR1 in Murine Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols
Murine tissue was stained as described elsewhere [66 (link)]. Tissue sections were prepared at 4-µm thickness. Immunohistochemical staining for ADAR1 (Santa Cruz Biotechnology, Dallas, TX, USA) was performed as previously described [66 (link)]. All slides were counterstained with hematoxylin. Slides were viewed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Jena, Germany).
Zhang X., Gao X., Hu J., Xie Y., Zuo Y., Xu H, & Zhu S. (2019). ADAR1p150 Forms a Complex with Dicer to Promote miRNA-222 Activity and Regulate PTEN Expression in CVB3-Induced Viral Myocarditis. International Journal of Molecular Sciences, 20(2), 407.
+ Open protocol
+ Expand
9

Silencing of Innate Immune Sensors

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were plated at 50-60% confluency in 60 mm plates (Corning) and incubated for 24 h. Cells were then transiently transfected using Lipofectamine RNAiMAX (Life Technologies) using two sets per gene of pooled siRNAs including ADAR1, cGAS, MAVS, MDA5 and IRF7, purchased from Santa Cruz Biotechnology and Dharmacon (Supplementary Table S1). Nonspecific scrambled siRNA from Dharmacon was used as control. After 48 hours of transfection, cells were lysed for immunoblotting analysis.
Sheikh T.N., Chen X., Xu X., McGuire J.T., Ingham M., Lu C, & Schwartz G.K. (2021). Growth Inhibition and Induction of Innate Immune Signaling of Chondrosarcomas with Epigenetic Inhibitors. Molecular cancer therapeutics.
+ Open protocol
+ Expand
10

Standard Western Blotting Techniques

Check if the same lab product or an alternative is used in the 5 most similar protocols
Standard methods were used for western blotting. Cells were lysed in lysis buffer and total protein contents were determined by the Bradford method. Thirty μg of proteins were separated by SDS–polyacrylamide gel electrophoresis under reducing conditions and blotted onto a polyvinylidene difluoride membrane (Millipore). Membranes were probed with specific antibodies. Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands visualized by chemoluminescence (Amersham Biosciences). All the primary antibodies were used at 1:1,000 dilution and secondary antibodies at 1:5,000 dilution. The following antibodies were used: Rabbit polyclonal Gabra3 (Santa Cruz Biotechnology), mouse monoclonal ADAR1 (Santa Cruz Biotechnology), Rabbit polyclonal AKT, pAKT, ERK, pERK, EGFR, pEGFR, N-Cadherin, ZO1 (Cell Signaling Technology), mouse monoclonal E-cadherin, Vimentin (Cell Signaling Technology), Fibronectin (Thermo Scientific), β-Catenin (BD Bioscience), β-actin (Sigma-Aldrich) and secondary peroxidase conjugated (GE healthcare). Uncropped scans of the immunoblots were shown in Supplementary Fig. 14.
Gumireddy K., Li A., Kossenkov A.V., Sakurai M., Yan J., Li Y., Xu H., Wang J., Zhang P.J., Zhang L., Showe L.C., Nishikura K, & Huang Q. (2016). The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis. Nature Communications, 7, 10715.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!