Whole-cell lysates from HTLV-1-infected and uninfected cells were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (ATTO, Tokyo, Japan). PVDF membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) and probed with anti-HBZ mAbs or anti-Histone H3 mAb (Cell Signaling Technology, Danvers, MA). PVDF membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary Abs (anti-rat IgG, HRP-linked whole Ab goat [NA935] or anti-mouse IgG, HRP-linked whole Ab sheep [NA931]; GE Healthcare, Buckinghamshire, England). After washing with TBS-T, the proteins were detected using ImmunoStar LD (Wako) and visualized with a C-DiGit® Blot Scanner (LI-COR Biosciences, Lincoln, NE). To enhance the western blotting signals, Western BLoT Immuno Booster (Takara Bio, Shiga, Japan) was used according to the manufacturer’s protocol.
Western blot immuno booster
Manufactured by Takara Bio
Sourced in Japan
The Western BLoT Immuno Booster is a laboratory product designed to enhance the sensitivity and performance of Western blotting techniques. It is a specialized reagent that assists in the visualization and detection of target proteins during the immunoblotting process.
Automatically generated - may contain errors
Lab products found in correlation
Western blot hyper hrp substrate, by Takara Bio (2 mentions)
C digit blot scanner, by LI COR (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Na k atpase, by Abcam (1 mentions)
Imagequant tl v8, by GE Healthcare (1 mentions)
Gapdh, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Las 4000, by Cytiva (1 mentions)
Western blot ultra sensitive hrp substrate, by Takara Bio (1 mentions)
Anti phospho smad1 5, by Cell Signaling Technology (1 mentions)
Las4000 mini, by Cytiva (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
5 protocols using western blot immuno booster
1
Western Blot Analysis of HTLV-1 HBZ Protein
2
Protein Extraction and Immunoblotting from Tobacco Leaves
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Protein extracts from tobacco leaves were prepared by grinding them in 5 volumes of buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM DTT, and Complete protease inhibitor cocktail (Roche Applied Science)]. Supernatants were cleared by centrifugation at 21,500×g for 15 min at 4 °C, and concentration of the protein extracts was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, USA) with bovine gamma-globulin as the standard.
For immunoblotting analyses, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck, Germany). After blocking with 5% nonfat dry milk, membranes were probed with 0.1 μg/ml anti-EAS antibody diluted with Western BLoT Immuno Booster (Takara, Japan) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody diluted with 1% nonfat dry milk at room temperature for 1 h. The antigen-antibody complexes were visualized using Western BLoT Hyper HRP Substrate (Takara, Japan).
For immunoblotting analyses, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck, Germany). After blocking with 5% nonfat dry milk, membranes were probed with 0.1 μg/ml anti-EAS antibody diluted with Western BLoT Immuno Booster (Takara, Japan) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled secondary antibody diluted with 1% nonfat dry milk at room temperature for 1 h. The antigen-antibody complexes were visualized using Western BLoT Hyper HRP Substrate (Takara, Japan).
3
Western Blot Analysis of Kidney Proteins
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Whole-kidney homogenates were adjusted to the same protein concentrations as determined by the DC Protein Assay (Bio-Rad, Hercules, CA, USA). Abs against the following proteins were used: UMOD (AbD Serotec), NKCC2 (Alpha Diagnostic International, San Antonio, TX, USA), Na+-K+-ATPase (Abcam), NCC (EMD Millipore), AQP2 (Abcam), arginine vasopressin (AVP) receptor 2 (Alomone Labs, Jerusalem, Israel), and GAPDH (MilliporeSigma, St. Louis, MO, USA). Abs were diluted with Western Blot Immuno Booster (Takara Bio). Immunoreaction was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA), and protein bands were scanned with an ImageQuant LAS 4000 Mini (GE Healthcare, Piscataway, NJ, USA) and quantified by using ImageQuant TL v.8.1 software (GE Healthcare). Information on the Abs used in this study is provided in Supplemental Table 1 .
4
Protein Expression Analysis of Cell Aggregates
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Cell aggregates treated with or without inhibitors from day 5 were dissociated using TrypLE Express Enzyme (Thermo Fisher Scientific) on day 6 and lysed in radioimmunoprecipitation assay buffer containing 50-mM Tris hydrochloride (pH 7.6), 150-mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease inhibitor cocktail (Nacalai) and ethylenediaminetetraacetic acid-free phosphatase inhibitor cocktail (Nacalai). A total of 2.7–9.0 μg of protein extracts were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membrane (Merck, Immobilon). Three biological replicates were analyzed. The following primary antibodies were used at the indicated dilutions: anti-beta-actin (rabbit, 1:1,000; Cell Signaling, 4970), anti-beta-catenin (rabbit, 1:1,000; Cell Signaling, 8480), anti-phospho-Smad1/5 (rabbit, 1:1,000; Cell Signaling, 9516), and anti-phospho-Smad2 (rabbit, 1:1,000; Cell Signaling, 3108). Anti-rabbit IgG, HRP-linked antibody (rabbit, 1:2,000; Cell Signaling, 7074) was used as a secondary antibody. The blocking buffer used was Western BLoT Immuno Booster (TaKaRa). The chemiluminescent substrate used was Western BLoT Ultra-Sensitive HRP Substrate (TaKaRa). The chemiluminescent signal was detected using LAS 4000mini (Cytiva) and analyzed using ImageJ.
5
Smad Signaling Pathway Protein Analysis
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hAECs were lysed with PROPREP (iNtRON Biotechnology, MA, USA) supplemented with Phosphatase Inhibitor Cocktail (EDTA free) (Nakarai tesque, Japan). Protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS–PAGE; Bio-Rad Laboratories, CA, USA) followed by a Western blot assay. EzBlockChemi (ATTO, Japan) was used for blocking, and Western BLoT Immuno Booster (Takara, Japan) was used for the reaction. The primary antibodies were as follows: Smad1, Smad2/3, phospho-Smad1 (Ser463/465)/5 (Ser463/465)/9 (Ser465/467), and phospho-Smad2 (Ser465/467)/3 (Ser423/425) (Cell Signaling Technology, MA, USA) at a 1:1,000 concentration, and GAPDH (GeneTex, CA, USA) at a 1:10,000 concentration. The secondary antibodies were peroxidase AffiniPure goat anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch Laboratory, PA, USA) at a 1:20,000 concentration. Western blot images were developed with Western BLoT Hyper HRP Substrate (Takara, Japan) and taken with a LumiCube System (Liponics, Japan).
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