The treated or untreated cells were collected, and the total protein was extracted by RIPA reagent (P0013B, Beyotime) with 1% protease inhibitors (P1030, Beyotime). After that, the concentration of the protein was determined by BCA kit (P0012, Beyotime). Then, cellular protein of 30 μg was separated by SDS-PAGE gel (P0012A, Beyotime). After electrophoresis for 90 min, the proteins were transferred on PVDF membranes (ISEQ00010/IPVH00010, MILLIPORE, MA, USA). Then, the membranes were blocked by 5% skimmed milk (D8340, Solarbio) for 1 h at room temperature, followed by incubation in primary antibodies (RUNX2, ab23981, 1：1,000, 60 kDa, Abcam, Cambridge, MA, USA; OCN, ab93876, 1：500, 11 kDa, Abcam; BCL2, ab182858, 1：2,000, 26 kDa, Abcam; GAPDH, ab181602, 1：10,000, 36 kDa, Abcam) at 4℃ overnight. Next day, the primary antibodies were recycled, and TBST (ST-673, Beyotime) was used to wash the membranes for 4 times (5 min for each time). Afterwards, secondary antibody (Goat Anti-Rabbit IgG H&L (HRP): ab6721, 1：10,000, Abcam) was incubated the membranes for 1 h (room temperature), followed by washing with TBST for 6 times (5 min for each time). Finally, ECL solution (WBKLS0500, MILLIPORE, Billerica, MA, USA) was dripped onto the membrane, and a specific imaging system (Bio-Rad, CA, USA) was used to visualize the band.
D8340
Manufactured by Solarbio
Sourced in China, United States
The D8340 is a laboratory equipment designed for conducting scientific analyses. It is a core component used in various research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Merck Group (4 mentions)
T1081, by Solarbio (2 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa reagent, by Beyotime (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Iseq00010 ipvh00010, by Merck Group (1 mentions)
St673, by Beyotime (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp ab6721, by Abcam (1 mentions)
Cd206, by Proteintech (1 mentions)
Foxo3a, by Proteintech (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Sds page electrophoresis solution, by Beyotime (1 mentions)
Trizol lysis buffer, by Thermo Fisher Scientific (1 mentions)
P0015, by Beyotime (1 mentions)
11 protocols using d8340
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis of Cell Lysates
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Cells were lysed in ice‐cold RIPA buffer containing protease inhibitor. The same amounts of protein were separated using an SDS‐PAGE gel and transferred to PVDF membranes (R1KB43643; Merck Co., Ltd.). The membranes were blocked in 5% skim milk (D8340; Solarbio Co., Ltd.) and then incubated overnight at 4°C with the primary antibodies for FoxO3a (66428‐1‐Ig; Proteintech Co., Ltd.), FoxO1 (18592‐1‐AP; Proteintech Co., Ltd.). Arg‐1 (66129‐1‐Ig; Proteintech Co., Ltd.), CD206 (60143‐1‐Ig; Proteintech Co., Ltd.), CD86 (13395‐1‐AP; Proteintech Co., Ltd.), GPX4 (A11243; ABclonnal Co., Ltd.), FTH1 (A19544; ABclonnal Co., Ltd.), HO‐1 (A19062; ABclonnal Co., Ltd.), LC3 (14600‐1‐AP; Proteintech Co., Ltd.), NCOA4 (A17330; ABclonnal Co., Ltd.), SLC7A11 (A2413; ABclonnal Co., Ltd.), β‐actin (HC201‐01; Transgen Co., Ltd.). Then subjected to HRP‐labelled secondary antibody for 1 h at room temperature. The reaction was visualised with chemiluminescent assay.
3
Comprehensive Protein Analysis Protocol
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The main reagents used in this study included TRIzol lysis buffer (Invitrogen, USA); reverse transcription kit: PrimeScript RT reagent kit with GDNA Eraser (RR 047A, Takara, Japan); qPCR kit: Tb Green Premix EX TAQ II (RR 820A, Takara); animal whole protein extraction kit (C510003, Sangon Biotech, China); BCA protein concentration assay kit (P0010, Beyotime Biotech, China); 5× loading buffer (P0015, Beyotime Biotech); SDS-PAGE gel preparation kit (P1200, Solar BioBeijing, China); SDS-PAGE electrophoresis solution (P00148, Beyotime Biotech); 10× EMT (D1060, Solar BioBeijing); methanol (CB/T693-1993, Shanghai Guangnuo Chemical Technology Co., Ltd., China); skimmed milk powder (D8340, Solar BioBeijing); 10×TBST buffer (powder) (T1087, Solar BioBeijing); nitrocellulose membrane (NC membrane) (37412133, PALL, USA); and ECL developer (34095, Thermo Fisher, USA).
4
Proteomic Analysis of Mouse Hippocampus
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The mouse hippocampus was lysed by radioimmunoprecipitation lysis buffer (Solarbio, Beijing, China, R0010) with a protease phosphatase inhibitor mixture (Solarbio, P1260). A BCA kit (Solarbio, PC0020) was applied to quantify the total protein concentration. Protein samples (40 μg per lane) were separated by 10% SDS-PAGE (Solarbio, P1200) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA, IPFL00010). The membranes were blocked with 5% skim milk (Solarbio, D8340) at room temperature (1 h) and subsequently immunoblotted with primary antibodies overnight at 4 °C: IL-33 (R&D, MND, USA, 1:500, AF3626), glial fibrillary acidic protein (GFAP) (Abcam, Cambridge, UK, 1:1000, ab7260), vesicular glutamate transporter 1 (vGlut1) (Abcam, 1:1000, ab227805), postsynaptic density protein-95 (PSD95) (Abcam, 1:1000, ab13552) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, 1:2000, ab8245). Next, 1X tris-buffered saline Tween (TBST) (Solarbio, T1081) was used to wash the membranes 3 times. They were then incubated with a secondary antibody at room temperature (40 min). Immunoreactivity was detected using enhanced chemiluminescence (Solarbio, P0018), and bands were measured using Image J analysis software (Version 1.50i, Rockville, MD, USA).
5
Protein Expression Analysis in Cells
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NP tissue and NP cells were lysed in the radioimmunoprecipitation assay (RIPA) buffer. The protein concentration was determined using a BCA kit (Thermo Scientific). 5% skim milk (Solarbio, D8340) was added to TBST (Solarbio, T1081) for 60 min to block membranes. After blocking, the membranes were incubated overnight at 4°C with the corresponding primary antibodies of Ace-tubulin (1:800; Cell Signaling Technology, 5335s), Collagen 2 (1:500; Proteintech, 28459-1-AP), SOX9 (1:1,000; Cell Signaling Technology, 82,630), Smad3 (1:1,000; Cell Signaling Technology, 9,523), YAP (1:1,000; Cell Signaling Technology, 14,074), p-YAP (1:1,000; Cell Signaling Technology, 13,008), TAZ (1:1,000; Cell Signaling Technology, 72,804), LATS1 (1:1,000; Cell Signaling Technology, 3,477), LATS2 (1:1,000; Cell Signaling Technology, 5,888), and GAPDH (1:1,000; Cell Signaling Technology, 5,174). Then, HP-linked secondary antibodies (1:5,000; Cell Signaling Technology, 7,074) were applied. The immunoblotting was detected by UVP ChemiDoc-It Imaging System (UVP, CA, USA) with the enhanced chemiluminescence detection kit (Thermo Fisher; 34,580) added to the membranes. Each blot was measured using ImageJ software for its integrated density.
6
Immunoprecipitation and Western Blot Analysis
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For immunoprecipitation, cells were washed with PBS for three times. Then, cells were lysed using NP-40 buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 20 mM β-glycerol phosphates, 1 mM Na orthovanadate, 20 mM NaF, 0.5% Nonidet P-40) supplemented with protease inhibitor for 30min at 4°C. Cell lysates were centrifuged for 20 min at 4°C, then the indicated antibodies and Protein G-Agarose (Roche, 11243233001) were added to the supernatants and incubate overnight at 4°C. After that, the suspensions were centrifuged at 3000 × g for 3 min at 4°C and then washed with PBS for three times. The sediments were suspended with 2× loading buffer and boiled for 8 min.
For western blot assay, the 2× loading buffer were added to cell lysates and boiled for 8 min. The samples were subjected to 10% SDS-PAGE and then transferred to PVDF (Polyvinylidene fluoride) membranes (Milipore, IPVH00010). The PVDF membranes were blocked with 5% skim milk (Solarbio D8340) for 1 h at room temperature (25°C) and then incubated with the indicated antibodies for 12h at 4°C. The PVDF membranes washed with TBS’T for three times and then incubated with the Goat anti-Mouse/Rabbit lgG-HRP secondary antibodies for 1h at 4°C. After being washed 3 times with TBS’T, the PVDF membranes were stained with ECL detection reagent (TIANGEN, PA112-01). The images were taken using Digital gel image analysis (TANON 5500).
For western blot assay, the 2× loading buffer were added to cell lysates and boiled for 8 min. The samples were subjected to 10% SDS-PAGE and then transferred to PVDF (Polyvinylidene fluoride) membranes (Milipore, IPVH00010). The PVDF membranes were blocked with 5% skim milk (Solarbio D8340) for 1 h at room temperature (25°C) and then incubated with the indicated antibodies for 12h at 4°C. The PVDF membranes washed with TBS’T for three times and then incubated with the Goat anti-Mouse/Rabbit lgG-HRP secondary antibodies for 1h at 4°C. After being washed 3 times with TBS’T, the PVDF membranes were stained with ECL detection reagent (TIANGEN, PA112-01). The images were taken using Digital gel image analysis (TANON 5500).
7
Quantification of Mitochondrial Protein Levels
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Hundred micrograms of protein per lane was subjected to 10% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk (D8340, Solarbio, Beijing, China) and incubated with primary antibodies against TOM70 (1:1000 dilution, ab89624, Abcam, Shanghai, China; 1:1000 dilution, 14528-1-AP, Proteintech, Wuhan, Hubei, China), TOM40 (1:2000 dilution,18409-1-AP, Proteintech, Wuhan, Hubei, China), TOM22 (1:500 dilution, 11278-1-AP, Proteintech, Wuhan, Hubei, China), TOM20 (1:2000 dilution, 11802-1-AP, Proteintech, Wuhan, Hubei, China), β-actin (1:2000 dilution, 20536-1-AP, Proteintech, Wuhan, Hubei, China), or GAPDH (1:50000 dilution, 60004-1-AP, Proteintech Wuhan, Hubei, China) overnight at 4°C, followed by incubation with secondary species-specific HRP-coupled antibodies (1:5000 dilution, E-AB-1034, Elabscience Wuhan, Hubei, China) for 1 h at room temperature. Proteins were visualized using an ECL chemiluminescence kit (WBKIS0100, Millipore, Darmstadt, Germany) and a chemiluminescence imaging system (ProteinSimple, ChenQ, CA, USA). Bands were quantified using ImageJ software.
8
Co60 Diet for Healthy Rat Feeding
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The Co60 irradiation maintenance diet (SWS9102, Xietong, Jiangsu, China) contains ≥50 g/kg of crude fiber, ≥40 g/kg of crude fat, ≤80 g/kg of ash, 6–12 g/kg of phosphorus, 10–18 g/kg of calcium, ≥180 g/kg of protein, ≥8.2 g/kg of lysine, ≥5.3 g/kg of methionine + cysteine and ≤100 g/kg of moisture. It is a standard balanced feeding pellet diet for rat feeding. This diet should keep them healthy, while eliminating the diet’s effects on blood pressure and blood chemistry. A total of 11% (w/v) skimmed milk from cow (D8340, Solarbio, Beijing, China) was prepared and sterilized at 105 °C for 15 min. A total 4 vol % each of the Lactiplantibacillus plantarum strains SR37-3 and SR61-2 was inoculated into sterilized skimmed milk respectively, cultured at 37 °C for 48 h and stored at −4 °C.
9
Western Blot Analysis of Proteins
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Total proteins from transfected AGS and MKN45 cells were isolated by RIPA buffer (R0010, Solarbio, Beijing, China) and quantified with the BCA protein quantification kit (ab102536, Abcam, Cambridge, UK) in line with the operating instructions. 20 μg protein samples were dissolved and electrically transferred onto a PVDF membrane (IPVH00010, EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk (D8340, Solarbio) at room temperature for 1 h, the membranes were incubated with primary antibodies against diverse proteins (RAD54B, 1:1000, ab168463; β-catenin, 1:500, ab16051; Axin, 1:1000, ab32197; cmyc, 1:20000, ab152146; MMP-7, 1:1000, ab216631; GAPDH, 1:2500, ab9485; all from Abcam) at 4°C overnight. Subsequently, the membranes were incubated with corresponding secondary antibodies for 2 h at room temperature and visualized by an ECL assay (P0018S, Beyotime, Shanghai, China). The band intensity was determined by ImageJ software (National Institutes of Health, USA).
10
Protein Extraction and Western Blot Analysis
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A radioimmunoprecipitation assay lysis buffer (Solarbio, R0030, Beijing, China) was used to extract total protein, which was then quantified using a bicinchnininc acid (BCA) assay kit (Solarbio, PC0020, Beijing, China). 20 µg of the extracted protein was transferred to polyvinylidene fluoride membranes (Millipore, IPVH00010, MA, USA). The membranes were blocked overnight at 4 °C using 5% skim milk powder (Solarbio, D8340, Beijing, China) and incubated with primary an-tibodies for 12 hours at a concentration of 1:1000. The antibodies used targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174), BCL2-Associated X (Bax) (2772), Bcl-2 (15071), NOD-like receptor thermal protein domain associated protein 3 (NLRP3) (15101), caspase-1 (83383), p-PI3K (17366), PI3K (4255), p-AKT (4060), AKT (9272), Nrf2 (33649), and HO-1 (43966), all obtained from CST (MA, USA). After primary antibody incubation, goat anti-rabbit IgG secondary antibody (1:1000, Solarbio, K1034G-AF594, Beijing, China) was applied for 1 hour. GAPDH served as the endogenous control. Finally, the grayscale values of each protein were analyzed using Image J software v1.5.24 (NIH, Bethesda, MD, USA).
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