To determine the relative amounts of TsxI, Hcp, and PilA, cells were incubated under the described conditions and harvested at indicated times. Harvested cells were washed and lysed by sonication in 0.2% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} TPM with protease inhibitor (Complete tablets, Mini EASYpack; Roche). Protein concentration for each sample was determined using a Bradford assay (Bio-Rad), and SDS-PAGE was performed with equal amounts of total protein loaded for each sample. Primary rabbit antibodies used were anti-FLAG (1:5,000; Sigma-Aldrich), anti-Hcp (1:1,000) (51 (link)), and anti-PilA (1:7,000) (35 (link)). For detection, horseradish peroxidase-conjugated goat anti-rabbit antibody was used (1:15,000; Pierce). Blots were developed using SuperSignal West Pico Plus chemiluminescent substrate (Thermo Scientific).
Mini easypack
Manufactured by Roche
Sourced in Germany
The Mini EASYpack is a compact and portable lab equipment designed for sample preparation. It provides a simple and efficient solution for sample homogenization and extraction. The device features a compact design and is suitable for use in various laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Complete tablet, by Roche (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Sep pak plus silica cartridges, by Waters Corporation (1 mentions)
Hrp conjugated anti rabbit igg secondary antibody, by Cell Signaling Technology (1 mentions)
Chemi lumi one, by Nacalai Tesque (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
G box chemiluminescence image capture system, by Syngene (1 mentions)
Bradford assay kit, by Beyotime (1 mentions)
Phosstop easypack, by Roche (1 mentions)
Phenylmethanesulfonyl fluoride, by Solarbio (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Precellys evolution homogenizer, by Bertin Technologies (1 mentions)
6 protocols using mini easypack
1
Quantifying Bacterial Virulence Factors
2
Western Blot Analysis of Hsp70 Protein
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Cell samples were lysed in RIPA buffer (radio-immunoprecipitation assay buffer: 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl; pH 8.0) and protease inhibitor (cOmplete tablets, Mini EASYpack, Roche Diagnostics, Mannheim, Germany) and separated by SDS-PAGE in 8% polyacrylamide gels. Samples were transferred to a nitrocellulose membrane by semi-dry blot. The membrane was then blocked with 5% milk (1 h at room temperature) and incubated with anti-Hsp70-antibody (HSP70 Polyclonal antibody, ProteinTech, Rosemont, IL, USA, 1:10,000 in 5% milk; 1.5 h at room temperature). After washing with Tris-buffered saline with Tween 20 (TBS-T), the membrane was incubated with a secondary HRP-conjugated antibody (1:1000 in 5% milk) for 1 h at room temperature. Protein expression of the target protein was normalized to β-Actin expression of the representative sample.
Western blots were developed with Clarity Western ECL Substrate (Biorad, Hercules, CA, USA), imaged via a fluorescence imaging system (PEQLAB fusion, PEQLAB, Erlangen, Germany) and quantified via ImageJ program (NIH).
Western blots were developed with Clarity Western ECL Substrate (Biorad, Hercules, CA, USA), imaged via a fluorescence imaging system (PEQLAB fusion, PEQLAB, Erlangen, Germany) and quantified via ImageJ program (NIH).
3
Liver Siphonaxanthin Quantification by HPLC
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Cells or tissue samples were homogenized in lysis buffer [20 mmol/L Tris-HCl, pH 8; 150 mmol/L NaCl, 1% Triton-X 100, protease inhibitor (cOmplete Tablets, mini EASYpack; Roche, Mannheim, Germany)]. The homogenate was centrifuged at 12,000 × g at 4 °C for 15 min to collect the supernatant. Protein concentration was determined using the DC protein assay kit (Bio-Rad Laboratories). Next, the proteins were separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Target proteins were probed with HMOX1 or β-actin primary antibody (1:1000; Cell Signaling, MA, USA) at 4 °C overnight, and then incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:2000, Cell signaling) at room temperature for 1 h. Signals were visualized with the substrate Chemi-lumi One (Nacalai Tesque) using a LAS-3000 visualizer (Fujifilm, Tokyo, Japan). Protein expression level was normalized using β-actin as an internal control.
2.9. Quantification of liver siphonaxanthin accumulation by high performance liquid chromatography Siphonaxanthin was extracted from the liver tissues and subjected to HPLC analysis as previously described [16] . The lipid extracts were loaded onto Sep-Pak Plus silica cartridges (Waters, MA, USA) to remove the TAG fraction, and dissolved in methanol for HPLC analysis.
The peak of siphonaxanthin was further confirmed from its characteristic UV spectrum.
2.9. Quantification of liver siphonaxanthin accumulation by high performance liquid chromatography Siphonaxanthin was extracted from the liver tissues and subjected to HPLC analysis as previously described [16] . The lipid extracts were loaded onto Sep-Pak Plus silica cartridges (Waters, MA, USA) to remove the TAG fraction, and dissolved in methanol for HPLC analysis.
The peak of siphonaxanthin was further confirmed from its characteristic UV spectrum.
4
Protein Extraction and Western Blot Analysis
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The protein samples were treated with ice‐cold radioimmunoprecipitation assay (RIPA) lysis buffer (CWBIO) with a protease inhibitor cocktail (cOmplete Tablets, Mini EASYpack, Roche) and a phosphatase inhibitor (PhosSTOP EASYpack, Roche) to extract total protein from cells and tissues. The concentration of protein was determined using a Bradford assay kit (Beyotime, Shanghai, China). Subsequently, 20 µg of protein from each sample was mixed with 5 × protein loading buffer and heated at 95–100 °C for 10 min. The denatured proteins were separated by electrophoresis on a 4–12% Bis‐Tris gradient precast gel (Tanon, Shanghai, China) and transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature. The relative intensity of the bands was detected using a chemiluminescent detection kit (Epizyme, Shanghai, China) and visualized with a G‐Box chemiluminescence image capture system (Syngene, Frederick, MD). The relative abundance of the target protein to GAPDH/ACTB was analyzed using ImageJ software and obtained as each target protein level. Primary antibodies are summarized in Table S7 (Supporting Information).
5
Western Blot Analysis of Epididymal Adipose Tissue
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The epididymal adipose tissue samples were homogenized in ice-cold lysis buffer containing complete tablets, mini EASYpack (Roche, Basel, Switzerland), and 1 mmol/L phenylmethanesulfonyl fluoride (Solarbio, Beijing, China) for 30 min. The homogenate was centrifuged at 4 °C with a speed of 12,000× g for 15 min and the supernatant was collected. Protein concentration was determined using the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Next, the proteins were separated using 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% skimmed milk in tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, then incubated with the following primary antibodies: β-actin (1:1000), p-ACC (1:1000), ACC (1:1000), p-HSL (1:1000), HSL (1:1000), PPARα (1:1000), and ATGL (1:1000) overnight at 4 °C. Subsequently, the membranes were rinsed using TBST three times and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 h at room temperature. The protein expression bands were detected using enhanced chemiluminescence (Easybio, Beijing, China) and visualized using a Tanon-4600SF chemiluminescence imager (Tanon Science & Technology Co., Ltd., Shanghai, China).
6
Quantifying sRANKL and OPG Expression
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The medial compartments of the right proximal tibia and distal femur were cut 5 mm from the joint line, weighed, and immediately stored at −80 °C. These samples were cut into small pieces with a bone cutter and crushed with extraction buffer, containing ice-cold phosphate-buffered saline (PBS), pH 7.4, protease inhibitors (cOmplete Tablets, Mini EASYpack, Roche) and 0.1% Triton X-100 using a Precellys Evolution homogenizer (Bertin Instruments, Rockville, MD, USA). The amount of added extraction buffer was determined by the weight of each sample to adjust the difference in sample volume. Soluble receptor activator of nuclear factor-kappa B ligand (sRANKL) and osteoprotegerin (OPG) expression levels were quantified and analysed using a sandwich ELISA kit for sRANKL and OPG (Rat sRANKL ELISA kit; Immundiagnostik AG, Rat Osteoprotegerin ELISA kit; Immundiagnostik AG, Bensheim, Germany) according to the manufacturer's protocols. The sRANKL/OPG expression ratio, which suggests increased osteoclast activity58 (link), was calculated.
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