McIlvaine buffer (Thermo Fisher Scientific) was adjusted to pH 4.0, 6.0, or 8.0 to examine the effect of pH. EGCG or nobiletin (final concentration, 3.0 mM) was added to SEA (final concentration, 5.0 µg/mL) diluted in McIlvaine buffer or PBS, and incubated at 37 °C for 24 h. Following centrifugation (4000× g, 5 min), the supernatant was collected. Milli-Q water was used as the control. Proteins were separated on acrylamide gels and transferred to a PVDF membrane, which was blocked with 5% skim milk powder (FUJIFILM Wako Pure Chemical Corporation). The PVDF membrane was soaked in rabbit anti-SEA (Sigma-Aldrich, St. Louis, MO, USA), diluted 1:1000 in PBS and incubated overnight at 4 °C. The PVDF membrane was washed with wash solution (20× Wash Solution Concentrate; KPL, Gaithersburg, MD, USA) three times for 5 min each with shaking at room temperature. The PVDF membrane was soaked in anti-mouse IgG (H + L) (KPL) diluted 1:10,000 in PBS at room temperature for 90 min with shaking. The membrane was washed with wash solution three times for 5 min each at room temperature with shaking. The PVDF membrane was then immersed in substrate solution (BCIP/NBT; KPL) and incubated at room temperature for 20 min to permit color development. The stained SEA bands were analyzed and quantified using ImageJ (National Institutes of Health, Bethesda, MA, USA).
Skim milk powder
Manufactured by Fujifilm
Sourced in Japan
Skim milk powder is a dry dairy product made by removing the fat content from milk. It serves as a key ingredient in various food and beverage applications, providing a source of protein, calcium, and other essential nutrients.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (2 mentions)
Mcilvaine buffer, by Thermo Fisher Scientific (1 mentions)
Trizma base, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Fujifilm (1 mentions)
Biotin labeling kit nh2, by Dojindo Laboratories (1 mentions)
Phosphate bufferedsaline pbs ph 7.4 solution, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin bsa, by Fujifilm (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
Temed, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Genview (1 mentions)
Acrylamide bis acrylamide, by Merck Group (1 mentions)
Tgx fastcast acrylamide gel, by Bio-Rad (1 mentions)
Prime western blotting detection reagent, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
10 protocols using skim milk powder
1
Evaluating EGCG and Nobiletin Effects on SEA
2
Extracting DNA from Environmental Samples
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Total DNA was extracted in duplicate from 0.5–1.0 g of samples using ISOIL for Bead Beating (Nippon Gene, Tokyo, Japan) with skim milk powder (Wako, Osaka, Japan) according to the manufacturer’s instructions with slight modifications (50 (link)). Additionally, for volcanic deposit samples from sites BR and BRU, DNA was extracted by the CTAB (hexadecyltrimethylammonium bromide) method (71 (link)) as described in our previous studies (16 (link), 17 (link), 23 (link)). Duplicates of extracted DNA were pooled together and subjected to molecular analyses.
3
Azide-Unit Functionalized Water-Soluble Photopolymer for Influenza Detection
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An azide-unit pendant water-soluble
photopolymer (AWP, 6%) was purchased from Toyo Gosei Co., Ltd. (Tokyo,
Japan). SA was purchased from ProSpec-Tany TechnoGene Ltd. (East Brunswick,
NJ). Bovine serum albumin (BSA), skim milk powder, hydrochloric acid,
and magnesium chloride hexahydrate were purchased from FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan). Trizma base and Tween 20
were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). Phosphate-buffered
saline (PBS; pH 7.4) solution and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)
phosphate (DDAO phosphate) diammonium salt were purchased from Thermo
Fisher Scientific, Inc. (Waltham, MA). Mouse anti-H5N1 HA monoclonal
antibody, recombinant H5N1 HA, recombinant H1N1 HA, recombinant H3N2
HA, recombinant H7N9 HA, and rabbit anti-avian influenza A HA polyclonal
antibody were purchased from Sino Biological, Inc. (Beijing, China).
DyLight 650-labeled goat antirabbit polyclonal antibody and alkaline
phosphatase-labeled goat antirabbit polyclonal antibody were purchased
from Abcam (Cambridge, MA). The biotin labeling kit-NH2 was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto,
Japan).
photopolymer (AWP, 6%) was purchased from Toyo Gosei Co., Ltd. (Tokyo,
Japan). SA was purchased from ProSpec-Tany TechnoGene Ltd. (East Brunswick,
NJ). Bovine serum albumin (BSA), skim milk powder, hydrochloric acid,
and magnesium chloride hexahydrate were purchased from FUJIFILM Wako
Pure Chemical Corporation (Osaka, Japan). Trizma base and Tween 20
were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). Phosphate-buffered
saline (PBS; pH 7.4) solution and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)
phosphate (DDAO phosphate) diammonium salt were purchased from Thermo
Fisher Scientific, Inc. (Waltham, MA). Mouse anti-H5N1 HA monoclonal
antibody, recombinant H5N1 HA, recombinant H1N1 HA, recombinant H3N2
HA, recombinant H7N9 HA, and rabbit anti-avian influenza A HA polyclonal
antibody were purchased from Sino Biological, Inc. (Beijing, China).
DyLight 650-labeled goat antirabbit polyclonal antibody and alkaline
phosphatase-labeled goat antirabbit polyclonal antibody were purchased
from Abcam (Cambridge, MA). The biotin labeling kit-NH2 was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto,
Japan).
4
Western Blot Reagent Specifications
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Agarose, Tween-20, Triton X-100, Ammonium Persulfate (AP), Acrylamide/Bis-acrylamide, 30%solution, TEMED and 4% paraformaldehyde were from Sigma-Aldrich Co. (St. Louis, MO, USA). Skim Milk Powder was purchased from FUJIFILM Wako Pure Chemical Corporation (Chuo-Ku, Osaka, Japan, 190–12,865). Protein sample Loading Buffers and PageRuler™ Prestained Protein Ladder were from Thermo Fisher Scientific (Waltham, MA, USA). DNA molecular weight markers were from Takara Bio, Inc. (Kusatsu, Shiga, Japan). Bovine Serum Albumin (BSA) was from Genview (Calimesa, CA, USA).
5
Suramin Effects on Cell Signaling Proteins
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Western blot analysis was performed on cells treated with different concentrations of suramin sodium (suramin) (Wako Pure Chemical Industries, Osaka, Japan) (0, 20, 50, and 100 μmol/L) for 24 hours in DMEM without FBS. The cells were homogenized in RIPA buffer (25 mmol/L Tris‐HCl, pH 7.6, 150 mmol/L NaCl, 1% NP‐40, 1% sodium deoxycholate, and 0.1% SDS) with protease inhibitor cocktail (p8340, Sigma). The protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Samples (20 μg) were separated on 10% TGX FastCast™ acrylamide gel (Bio‐Rad) and transferred to a Amasham™ Hybond™ 0.2 μm polyvinylidene fluoride (PVDF) membrane (GE Healthcare). Blocking of the membrane was performed using PBS with 5% skim milk powder (Wako Pure Chemical Industries). The antibodies used in this study were specific to HuR (sc‐5261, 1:2000, Santa Cruz, Santa Cruz, CA, USA), cyclin A (611268, 1:2000), cyclin B1 (554178, 1:2000), Cox2 (610203, 1:2000, BD Biosciences, Cambridge, MA, USA), and β‐actin (A5441, 1:10 000, Sigma). The secondary antibody was horseradish peroxidase‐conjugated IgG (Jackson ImmunoResearch Laboratories, 1:5000‐10 000, West Grove, PA, USA). The protein bands were visualized with the Amersham™ ECL™ start or Prime Western Blotting Detection Reagent (GE Healthcare) and detected by LAS‐4000 mini (GE Healthcare).
6
Preparation of Lipid Nanoparticles for Cell Culture
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Fetal bovine serum (FBS) was obtained from Biosera (East Sussex, UK). Sodium phosphotungstate and 4% paraformaldehyde phosphate buffer solution (PFA), and 30% (w/v)-acrylamide/bis mixed solutions were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Phosphatidylcholine (PC), cholesterol, and phosphatidylserine (PS) were purchased from Nippon Fine Chemical Co. Ltd. (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Actinomycin D, penicillin-streptomycin-L-glutamine solution (×100) (PSG), polyoxyethylene (20) sorbitan monolaurate (Tween 20), sodium dodecyl sulfate (SDS), and skim milk powder were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals used were of the highest commercially available grade.
7
Protein Extraction and Western Blot
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We extracted total proteins from cells by RIPA lysis buffer (FUJIFILM Wako Pure Chemical Inc) and detected their concentration by protein assay BCA kit (FUJIFILM). The proteins were separated by SDS polyacrylamide gel electrophoresis and moved to a polyvinylidene fluoride membrane. We added 5% skim milk powder (FUJIFILM) in 1 × TBST to block the membrane for 1.5 h, then incubated the membrane overnight with specific primary antibodies at 4 °C. Among them, BIRC5 (Survivin) antibody was purchased from Proteintech Japan; antibodies for PARP, cleaved PARP, caspase3, and β-actin were purchased from Cell Signaling Technology. The next day we washed the membrane three times with 1 × TBST and incubated with HRP conjugated anti-rabbit or anti-mouse secondary antibodies (Southern Biotech) for 1.5 h. Finally, the protein was detected by an Amersham Imager 600 (Thermo).
8
Western Blot Analysis of Puromycin Incorporation
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Western blotting was conducted as previously reported(Rashad et al., 2022 (link); Rashad et al., 2020a (link)). Briefly, for the analysis of puromycin incorporation assay, cells were incubated in a complete growth medium with 10 μg/ml puromycin then homogenized in T-PER™ tissue protein extraction reagent (Thermo Fisher Scientific, Cat# 78510) with Triton(R) X-100 (Nacalai Tesque, Cat# 35501-15) and cOmplete™ protease inhibitor cocktail (Roche, Cat# 4693116001). Proteins were isolated from the supernatant, quantified using the Brachidonic-acid assay kit (BCA) (Thermo Fisher Scientific, Cat# 23227), separated on Mini-PROTEAN TGX gel (Bio-Rad, Cat# 4561096), and transferred to Polyvinylidene difluoride membrane (PVDF) (Bio-Rad, Cat# 1704156). Membranes were blocked in 5% Skim milk powder (Fujifilm, Cat# 190-12865) with Phosphate buffered saline with tween® (PBS-T) (Takara, Cat# T9183). The primary antibody was incubated overnight at 4°C, followed by room temperature incubation with the secondary antibody (IgG detector solution v2). Signal detection was performed using Pierce ECL substrate reagent (Thermo Fisher Scientific, Cat# 32106) on a ChemiDoc machine (Bio-Rad). To normalize protein expression, membranes were stripped with Restore™ stripping buffer (Thermo Fisher Scientific, Cat# 21059) and reprobed with Anti-beta actin antibody.
List of antibodies used:
List of antibodies used:
9
Histone Modification Analysis by Western Blot
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RIPA buffer and PMSF (100:1) (Solarbio; Beijing, China; No. R0010) were added to the cells, which were then lysed for 40 min on ice. After centrifugation at 12,000 × g for 15 min at 4°C the supernatant was collected. Protein concentrations were determined using a Bicinchoninicacid (BCA ) protein concentration assay kit. Radioimmunoprecipitation assay (RIPA ) buffer and 6 × SDS loading buffer (Beyotime, China; No. P0015F) was added, and the protein samples were denatured at 100°C for 30 min. The samples were electrophoresed at 80 V for 10 min, followed by 120 V for 30 min. Then, 5% skim milk powder (Wako, China; No. 190-12865) was placed on a shaker at room temperature and incubated for 2 h. Antibody was added to the incubation container, shaken for 1 h and incubated overnight at 4°C(H3K4me2, 1:2,000; Histone H3, 1:2,000; Goat Anti‐Rabbit IgG, 1:2,000; β‐actin, 1:100,000; TDRD1, 1:2,000). Secondary antibody was then added and incubated for 2 h on a shaker at room temperature. ECL chromogenic solution (Vazyme, China; No. E412-01) was added to the surface, and images were acquired.
10
Western Blot Analysis of Cell Signaling Proteins
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Total cell lysates were loaded onto 7.5% agarose gel. Electrophoresis was run and wet transferred to PVDF membrane (Millipore) at 300 mA for 2 hr. After blocking with 5% skim milk powder (Wako) in T-TBS for 1 hr, membranes were incubated with primary antibody solutions at 4°C overnight. Membranes were then washed with T-TBS, and incubated with horseradish peroxidase conjugated anti-IgG antibodies (GE Healthcare) at RT for 1 hr. Antibody binding was detected with a chemiluminescent substrate (Pierce ECL Plus Western Blotting Substrate, Thermo Fisher Scientific) and X-ray film (GE Healthcare). Antibodies used were:rabbit anti-Bach1 polyclonal antibody (1:1000dil) [18 (link)], rabbit anti-FoxO1 monoclonal antibody (1:1000 dil, 2880, Cell Signaling), rabbit anti-phospho-FoxO1 polyclonal antibody (1:1000 dil, 9461, Cell Signaling), rabbit anti-Smad2/3 monoclonal antibody (1:300 dil, 8685, Cell Signaling), rabbit anti-phospho-Smad2/3 monoclonal antibody (1:200 dil, 8828, Cell Signaling) and mouse anti-GAPDH monoclonal antibody (1:5000 dil, ab8245, Abcam).
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