Protein extraction from cell lines was performed using RIPA buffer (5 nM EDTA, 150 mM NaCl, 1% Triton, 20 nM Tris pH 8 and 1:200 protein inhibitors). After Bradford colorimetric protein quantification samples were run on a SDS/PAGE acrylamide gel for 2 h and transferred to a PVDF membrane. Membranes were blocked for 1 h with 5% milk and incubated with the primary antibodies described above overnight and with the secondary antibodies -Goat anti-rabbit (Ref. P0448, dilution 1:2000, Agilent; Santa Clara, CA, USA) and rabbit anti-mouse (Ref. P0260, dilution 1:2000, Agilent; Santa Clara, CA, USA)- for 1 h at room temperature. Membranes were developed using Immobilon Immobilon Western Chemiluminiscent (Ref. WBKLS0100, Merck Millipore; Billerica, MA, USA). Membranes were finally stained with naphtol blue (Ref. N3393, Sigma-Aldrich; St. Louis, MO, USA) to detect all proteins transferred to the membranes in order to normalize the ADAR protein bands. The following antibodies were tested: ADAR1 (Ref. AMAb90535, dilution 1:100, Sigma-Aldrich; St. Louis, MO, USA) and ADAR2 (Ref. sc-73409, dilution 1:50, Santa Cruz; Heidelberg, Germany, EU).
Sc 73409
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
Sc-73409 is a laboratory instrument designed for performing various analytical and experimental tasks. It is a versatile piece of equipment that can be utilized in a wide range of scientific applications. The core function of Sc-73409 is to facilitate reliable and accurate data collection and analysis. However, a more detailed description of its specific features and intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Amab90535, by Merck Group (2 mentions)
α gapdh, by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Coomassie plus bradford reagent, by Thermo Fisher Scientific (1 mentions)
Sc 73408, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Envision secondary antibody, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
4 protocols using sc 73409
1
Western Blot Analysis of ADAR Proteins
2
Western Blot Analysis of ADAR Proteins
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Transduced cells were lysed using M-PER lysis buffer with 5 mM EDTA and HALT protease inhibitor cocktail (all Thermo Scientific) and cleared by centrifugation at 14,000g at 4°C for 10 min. Protein concentrations were determined using Coomassie Plus Bradford reagent (Thermo Scientific) and equal amounts of protein were prepared using Laemmli buffer (Bio-Rad) with added DTT. After denaturation at 95°C for 5 min, proteins were separated on 4%–20% Mini-Protean TGX gels (Bio-Rad) and blotted onto PVDF membranes using the TransBlot Turbo Transfer system (Bio-Rad). Membranes were blocked with blocking buffer (5% milk, 5% FBS, 1% BSA, 1 M Glycine) at room temperature and probed with primary antibodies at 4°C overnight. Primary antibodies were α-ADAR1 (sc-73408, Santa Cruz Biotechnology; 1:500), α-ADAR2 (sc-73409, Santa Cruz Biotechnology; 1:1000), and α-GAPDH (MAB374, Millipore; 1:25,000). Membranes were probed with α-mouse HRP secondary antibody (P0447, DAKO, Glostrup, Denmark) for 1 h at room temperature, and chemiluminescence was detected using Immobilon Forte Western HRP substrate (Millipore) on an Amersham Imager 600 (GE Healthcare).
3
Immunohistochemical Analysis of ADAR2 and BAP1
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TMA construction was achieved as previously described [29 (link)]. Immunohistochemistry was performed as previously described [30 (link)]. For ADAR2 immunostaining, we used rabbit anti‐ADAR2 (Santa Cruz Biotechnology Cat# sc‐73409, RRID:AB_2289194) antibody. TMA spots with a lack of tumor tissue were excluded from the analysis, resulting in the analysis of 178 patients' samples. Immunohistochemical evaluation of the TMAs was conducted by two independent observers in a blinded manner. The average H‐score of all the cores obtained from the same tumor specimen was determined by the percentage of cells having nuclear ADAR2 positivity and scored as 0 (0%), 1 (1–9%), 2 (10–49%), or 3 (50% and more). BAP1 staining was performed as previously described [29 (link)] and BAP1 immunohistochemistry data was stratified into four groups: nuclear only, cytoplasmic only, absent, and combination of nuclear and cytoplasmic as recently described [23 (link)].
4
Immunohistochemical Analysis of ADAR1 and ADAR2 in Endometrial Tissue
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A total of three TMAs were constructed as described previously47 (link). Paraffin-embedded TMA or individual sections of the samples were mounted on slides, deparaffined and rehydrateted. Antigen retrieval was done during 4 min at 115 °C (pH 6) and subsequently blocked with peroxidase (3%) for 5 min and incubated with the primary antibody for 1 h and 30 min at room temperature in a humidified chamber. After blocking for 30 min with EnVision secondary antibody (Ref. K5007-100 ml, Agilent; Santa Clara, CA, USA) slides were treated with DAB (Ref. K3468, Agilent; Santa Clara, CA, USA) reagent and counterstained with hematoxylin for 30 sec. Finally, a dehytratation step and DPX mounting were performed. Pictures were taken using the Olympus BX41 microscope (20X). The following antibodies were tested: ADAR1 (Ref. AMAb90535, dilution 1:100, Sigma-Aldrich; St. Louis, MO, USA) and ADAR2 (Ref. sc-73409, dilution 1:50, Santa Cruz; Heidelberg, Germany, EU). A pathologist evaluated the expression using two criteria: the intensity of staining (0, no staining; 1, weak intensity; 2, moderate intensity; 3, high intensity) and the percentage of endometrial epithelial stained cells (0–100). The product of the two scores yielded final values on a sacale ranging from 0 to 300.
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