Cell lysates were prepared by 1% Triton X-100 and cell debris was removed by centrifugation. Cell lysates were boiled in sodium dodecyl sulfate sample buffer with a reducing reagent (Nacalai Tesque, Inc.). These proteins (10 µg) were electrophoresed on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 10 µg/ml of C20Mab-11, 1 µg/ml of NZ-1 (anti-PA tag) or 1 µg/ml of anti-β-actin for control (clone AC-15; Sigma-Aldrich Corp.), followed by incubation with secondary antibody peroxidase-conjugated anti-mouse immunoglobulin (1:1,000; Agilent Technologies Inc., Santa Clara, CA, USA) or anti-rat IgG (1:10,000; Sigma-Aldrich Corp.), respectively. Finally, proteins were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co. Ltd.).
Reducing reagent
Manufactured by Nacalai Tesque
Sourced in Japan
Reducing reagent is a laboratory chemical used to facilitate reduction reactions. It is a core tool for researchers and scientists working in various fields of chemistry and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Skim milk, by Nacalai Tesque (1 mentions)
Polyacrylamide gel, by Fujifilm (1 mentions)
Anti rat igg, by Merck Group (1 mentions)
Peroxidase conjugated anti mouse immunoglobulin, by Agilent Technologies (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Af7646, by Cell Signaling Technology (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Anti hif 2α, by Novus Biologicals (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using reducing reagent
1
Western Blot Analysis of Protein Samples
2
Extraction and Analysis of Root Proteins
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Root proteins were extracted using the buffer [50 mM potassium phosphate buffer (pH 7.0), 200 mM sodium chloride, 10 mM EDTA, 0.1% (v/v) Triton-X100, 0.1% (v/v) N-lauroylsarcosine sodium salt] including 10 mM 2-mercaptoethanol. After vigorous mixing, the mixture was centrifugated at 20,700×g for 15 min at 4°C, and the supernatant was collected. Proteins in the root extracts were quantified using the Bradford method.27 (link)) Samples for SDS-PAGE were prepared by adding a sample buffer solution containing a reducing reagent (Nacalai Tesque, Inc., Kyoto, Japan) to 10 µg of root proteins and 10 µL of xylem sap and then heating them in a water bath at 98°C for 5 min. Proteins were separated using a 15% polyacrylamide gel and transferred to a PVDF-Plus membrane (Maine Manufacturing, LLC, Sanford, ME, USA) to detect the MLPs using anti-MLP-PG1 and anti-MLP-GR3 antibodies.9 )
3
Immunoprecipitation of SALL3 and DNMT3B
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Cells were lysed for 30 min at 4 °C in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) containing complete mini EDTA-free protease inhibitor cocktail (Roche). Cell lysates were clarified by centrifugation for 30 min at 20,000 × g at 4 °C. Cleared lysates were then incubated with antibody for 2 h at 4 °C with rotation. The following antibodies were used: anti-SALL3 (Abnova PAB28233, 1:100), anti-DNMT3B (R&D Systems AF7646, 1:100), rabbit IgG (Cell Signaling Technology 2729, 1:100), and sheep IgG (R&D Systems 5-100-A, 1:100). The lysates were then incubated with protein G magnetic beads (Life Technologies) with rotation for 30 min at 4 °C. The beads were washed three times with lysis buffer. Proteins bound to the beads were eluted using sodium dodecyl sulfate (SDS) sample buffer solution with reducing reagent (Nacalai) at 95 °C for 5 min.
4
Western Blot Analysis of HIF-1α and HIF-2α
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ARPE-19 and fhRPE cells were lysed on ice in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) to extract the cellular proteins. Equal amounts of protein (based on the BCA assay) were treated with a sample buffer solution containing a reducing reagent (Nacalai Tesque, Inc., Kyoto, Japan). The samples were heated to 95°C for 3 min, fractionated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and immunoblotted. Anti-β-actin antibody (1 : 5000, Cat #3700, Cell Signaling Technology, Danvers, MA, USA) was used as an internal standard to normalize each sample. Anti-HIF-1α (1 : 1000, Cat #36169, Cell Signaling Technology) and anti-HIF-2α (1 : 1000, Cat #NB100-122, Novus Biologicals, Centennial, CO, USA) were used as primary antibodies. The immunoblots were developed using horseradish peroxidase-conjugated secondary antibodies (1 : 5000; GE Healthcare, Princeton, NJ, USA). Signals were detected using an ECL kit (Ez WestLumi plus, ATTO, Tokyo, Japan) and imaged using chemiluminescence (ImageQuant™ LAS 4000 mini, GE Healthcare). All raw data of western blotting is available in Supplement Figure 1 .
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