Skeletal muscle fibers were isolated from adult Wistar rats. Animals were anesthetized and sacrificed by procedures that are in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003) and approved by the Institutional Animal Care and Use Committee (IACUC) of College of Medicine, National Taiwan University. In brief, vastus lateralis (thigh muscle) was dissected and homogenized in the T-PER tissue extraction reagent (Thermo Scientific; 0.1 g muscle per 500 μl reagent). Lysates were cleared by micro-centrifugation at 13,000 rpm for 15 min. The supernatants were pre-cleared with protein A sepharose beads (GE Healthcare Biosciences) for 2 hrs, incubated with the anti-CLC-1 antibody at 4 °C for 2 hrs, and then incubated with protein A sepharose beads at 4 °C for 16 hrs. Beads were gently spun down and washed twice in the T-PER reagent and then three times with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer. Where indicated, the specificity of immunoprecipitation was verified with mouse IgG (Sigma).
T per tissue extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
T-PER tissue extraction reagent is a buffer solution used for the extraction of proteins from tissue samples. It is designed to efficiently solubilize and extract proteins from a variety of tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Phosphatase inhibitor cocktail, by Selleck Chemicals (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Peroxidase conjugated goat anti rabbit immunoglobulin igg h l, by Proteintech (2 mentions)
Supersignal west pico plus substrate kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Met clone sp44, by Roche (1 mentions)
Powerplex 16 system, by Promega (1 mentions)
Enhanced chemiluminescence detection system, by GE Healthcare (1 mentions)
Pparγ, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
12 protocols using t per tissue extraction reagent
1
Isolation and Immunoprecipitation of Rat Skeletal Muscle CLC-1
2
Whole-Cell Lysate Immunoblotting Protocol
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Whole-cell lysates were resolved using T-PER Tissue Extraction Reagent (Thermo Fisher Scientific Inc., MA, USA) with a complete ethylenediaminetetraacetic acid-free protease inhibitor and phosphatase inhibitor cocktail (Selleck Chemicals). Immunoblotting was performed using a standard method. The detailed information of the antibodies used in this study is summarized in Supplementary Table 7 . The quality of the gel loading and the transfer processes were assessed by immunostaining the blots with the Vinculin antibody.
3
Cell Line Authentication and Western Blotting
4
Western Blot Analysis of Liver Proteins
5
Western Blot Analysis of MOG from Liver
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Protein was extracted from liver tissue using T-PER Tissue Extraction Reagent (Thermo) in the presence of Halt Protease Inhibitor (Thermo). Total protein concentration was measured using the bicinchoninic acid protein assay (Pierce). Samples was separated on 4%–20% Mini-PROTEAN TGX gels (Bio-Rad) and transferred to polyvinylidene fluoride (PVDF) membrane following standard protocols. After blocking, the membrane was incubated for 1 hr at room temperature with antibody against MOG or β-actin in 1% fat-free dry milk in 1× tris-buffered saline with tween (TBST). HRP-conjugated secondary antibody was used for signal detection with the ECL 2 Western Blotting Substrate (Pierce).
6
Molecular Mechanisms of CdCl2-Induced Toxicity
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CdCl2 was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), with a purity of >98.0%. Alanine/aspartate aminotransferase (ALT/AST) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). T-PER tissue extraction reagent, NE-PER nuclear and cytoplasmic extraction reagents, SuperSignal West Pico PLUS Substrate kit, and Pierce BCA Protein Assay Kit were obtained from ThermoFisher Scientific (Waltham, MA, USA). Murine IL-1β and IL-6 ELISA kits were purchased from Neobioscience (Shenzhen, China). The antibodies that were used for immunoblotting anti-Keap1, -p38, -phospho-p38, -ERK, -phospho-ERK, -JNK, -phospho-JNK, -NLRP3, -NF-κB p65, and -phospho- NF-κB p65 were purchased from Cell Signaling Technology (Danvers, MA, USA) (1:1000 dilutions). The antibodies against Nrf2 and HO-1 were all purchased from Santa Cruz (Santa Cruz, CA, USA) (all 1:200 dilutions). Antibodies against GAPDH were purchased from Abways Technology (Shanghai, China) (1:2000 dilutions). Peroxidase-conjugated goat anti-rabbit immunoglobulin IgG (H + L), anti-mouse IgG (H + L), and anti-goat IgG (H + L) were obtained from Proteintech Group (Wuhan, China). Other reagents, unless indicated, were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
7
RANKL and HX112 Stimulation Reveals Signaling Pathways
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Total protein of RAW264.7 cells stimulated with RANKL and HX112 for either 30 min or 24 h was obtained using the T-PER tissue extraction reagent (Thermo Fisher, USA). Whole lysates were separated on Bolt™ Bis-Tris Plus Gels (Invitrogen, USA) and transferred to polyvinylidene fluoride (Cytiva, Korea). Then, membrane was blocked with 1X TBS Blocking buffer (Thermo Fisher, USA) and then incubated with a specific antibodies for NFATc1 (1:1000, Abcam, UK), c-FOS, p-Src, Src, p-PI3K, PI3K, p-AKT, AKT, p-JNK, JNK, p-p38, p38, p-ERK, ERK (1:1000, Cell Signaling, USA), and β-actin (1:5000, Sigma). Subsequently, HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (1:5000, Cell Signaling) were additionally treated, and the target proteins were identified by enhanced chemiluminescence solution (Thermo Fisher) and ChemiDoc MP imaging system (Bio-Rad, USA).
8
Western Blot Analysis of Testicular Proteins
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Finely chopped testis samples were obtained using a Potter−Elvehjem homogenizer apparatus on ice, and whole cell proteins were extracted using T-PER tissue extraction reagent (Thermo Scientific Inc., Hanover Park, IL, USA). Protease inhibitor (1:200 dilution; Sigma-Aldrich) was incorporated to avoid protein degradation. Wester blotting procedure was prepared according to the method previously described by Moreno-Fernandez et al.49 (link). The blots were incubated with NRF2 polyclonal (Abcam, UK; dilution 1:500), PGC-1α monoclonal (Merck., Darmstadt, Germany; dilution 1:1000) and mouse anti-β-actin monoclonal (Abcam, UK; dilution 1:1000) as primary antibodies, in 5% dry milk in TTBS overnight at 4 °C with shaking. β-Actin was used as loading control. The bands were visualized with Luminata forte western HRP substrate (Merck KGaA, Darmstadt, Germany). Signal quantification and recording densitometry of each band were performed with chemiluminescence in ImageQuant LAS 4000 (Fujifilm Life Science Corp., USA). All results were analyzed with ImageJ software. Measurements in duplicate were used to determine intra-assay variability.
9
Immunoprecipitation of ClC-2 from Murine Testis
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C57BL/6 mice were handled in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996, Bethesda, MD, USA). All procedures involving animals were performed in conformity with the animal protocol approved by the Institutional Animal Care and Use Committee (IACUC), College of Medicine, National Taiwan University.
Testes dissected from mice (about 6-weeks-old; weighing about 19 g) were homogenized in ice-cold T-PER tissue extraction reagent (Thermo Scientific, Waltham, MA, USA; 1 testis per 400 µL) containing protease inhibitor cocktail. Lysates were cleared by micro-centrifugation at 13,360× g for 15 min. Solubilized lysates were pre-cleared with protein G sepharose beads (GE Healthcare Biosciences, Piscataway Township, NJ, USA) for 2 h at 4 °C, and then incubated for 16 h at 4 °C with protein G sepharose beads pre-coated with rabbit IgG or rabbit anti-ClC-2 antibody. Beads were gently spun down and washed twice in a wash buffer [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO3, 20 Tris-HCl, pH 7.5] supplemented with 0.1% Triton X-100, and then twice with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer.
Testes dissected from mice (about 6-weeks-old; weighing about 19 g) were homogenized in ice-cold T-PER tissue extraction reagent (Thermo Scientific, Waltham, MA, USA; 1 testis per 400 µL) containing protease inhibitor cocktail. Lysates were cleared by micro-centrifugation at 13,360× g for 15 min. Solubilized lysates were pre-cleared with protein G sepharose beads (GE Healthcare Biosciences, Piscataway Township, NJ, USA) for 2 h at 4 °C, and then incubated for 16 h at 4 °C with protein G sepharose beads pre-coated with rabbit IgG or rabbit anti-ClC-2 antibody. Beads were gently spun down and washed twice in a wash buffer [(in mM) 100 NaCl, 4 KCl, 2.5 EDTA, 20 NaHCO3, 20 Tris-HCl, pH 7.5] supplemented with 0.1% Triton X-100, and then twice with the wash buffer. The immune complexes were eluted from the beads by heating at 70 °C for 5 min in the Laemmli sample buffer.
10
Immunoblotting Analysis of Protein Signaling
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Whole cell lysates were resolved using T-PER Tissue Extraction Reagent (Thermo Fisher Scientific Inc., MA, USA) with a complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor and phosphatase inhibitor cocktail (Selleck Chemicals). Immunoblotting was performed using a standard method. The following primary antibodies and dilutions were used: anti-human p110α (#4249, 1:1000), anti-human p-AKT(Ser473) (#4060, 1:1000), anti-human AKT (#4691, 1:1000), anti-human p85 (#4257, 1:1000), anti-human ERK1/2 (#4695, 1:1000), anti-human p-ERK1/2 (#4370, 1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#5174, 1:2000) antibodies from Cell Signaling Technology and anti-HA antibody (#H3663, 1:1000) from Sigma-Aldrich. The quality of the gel loading and the transfer processes was assessed by immunostaining the blots with the GAPDH antibody. The densitometry analysis was performed using ImageJ software (NIH, Bethesda, MD). Uncropped scans of important blots are provided as Supplementary Fig. 13 in the Supplementary Information.
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