The BATs were homogenized in RIPA buffer (P0013B, Beyotime, Beijing, China) supplemented with 1:100 protease inhibitors (P1005, Beyotime, Beijing, China) and centrifuged at 14,000 g , 10 min. Protein concentrations were adjusted with BCA assay (P0012, Beyotime, Beijing, China), denatured in SDS sampling buffer (95°C, 5 min) and then subjected to 12% SDS-PAGE. Protein samples were then electrotransferred to a polyvinylidene fluoride membrane (Millipore Corp.) for 2 h. After blocking with 5% fetal bovine serum (FBS) for 2 h at room temperature, membranes were incubated with primary antibodies: UCP-1 (ab10983, 1:2000; Abcam); β-actin (#4967, 1:4000 CST, Danvers, MA, USA) at 4°C overnight and with secondary antibodies (goat anti-rabbit IgG H&L (HRP (ab6721, Abcam, 1:2000)) at room temperature for 1 h. Bands were visualized with Immobilon Western chemiluminescent substrate (Millipore, cat. no. WBKLS0100, 200 µL) and UVP 810 gel-imager (UVP, Upland, CA, USA), and the intensities were analyzed using Image J software (NIH, USA).
Ucp1 ab10983
Manufactured by Abcam
Sourced in United States
UCP1 (ab10983) is a protein-coding gene that plays a role in thermogenesis. This antibody can be used to detect UCP1 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
Immobilon western chemiluminescent substrate, by Merck Group (1 mentions)
P1005, by Beyotime (1 mentions)
Bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Fabp4 e71703 98, by Huabio (1 mentions)
Cocktail 45 8099, by Thermo Fisher Scientific (1 mentions)
Phospho stat1 tyr701 58d6, by Cell Signaling Technology (1 mentions)
Sodium palmitate p9767, by Merck Group (1 mentions)
4 hydroxytamoxifen 4 oht, by Bio-Techne (1 mentions)
Uk5099, by Bio-Techne (1 mentions)
Prdm16 ab106410, by Abcam (1 mentions)
High density lipoprotein cholesterol hdl c, by Nanjing Jiancheng (1 mentions)
Low density lipoprotein cholesterol ldl c, by Nanjing Jiancheng (1 mentions)
Reverse transcription kit, by Sangon (1 mentions)
Trizol reagent, by Sangon (1 mentions)
6 protocols using ucp1 ab10983
1
Western Blot Analysis of UCP-1 Protein
2
Adipose Tissue Protein Analysis
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Small pieces of adipose tissues were homogenized with lysis buffer. Total protein concentrations were determined by Bradford protein assay kit (Thermo Scientific, United States). Equal amounts of protein samples were loaded. Proteins were separated via 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Millipore, Billerica, MA, United States). Membranes were blocked in 5% skim milk for 60 min, Primary antibody (UCP-1, ab10983, Abcam, 1:2000 dilution; PGC-1α, ET1702-96, HUABIO, 1:2000 dilution) was then incubated overnight at 4°C. The next day membranes were washed and incubated in secondary antibody for 1 h. The target bands were detected by ECL chromogenic substrate. Protein bands were quantified using ImageJ software. All representative images were repeated in three independent experiments.
3
Protein Isolation and Western Blot Analysis
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Total protein was isolated from cells or tissues using RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined with the BCA protein assay (Thermo Fisher Scientific, USA). Protein separation and western blot analysis were conducted as described earlier [5 (link)]. GADD45A antibody (sc-6850, 1: 1,000) was from Santa Cruz Biotechnology (Santa Cruz). Perilipin-1 (ab61682, 1:2000) and UCP1 (ab10983, 1:1000) were from Abcam. FABP4 (E71703-98, 1:2000) and GAPDH (EM1101, 1:5000) were from HuaBio. Cocktail (45-8099, 1:2000) was from Thermo Fisher Scientific. Stat1 (D4Y6Z, 1: 1000) and Phospho-Stat1 (Tyr701) (58D6, 1:1000) were obtained from Cell Signaling Technology. The horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG, 111-035-003 or anti-mouse IgG; 115-035-003, Jackson ImmunoResearch) was diluted 1:10,000. Immunodetection was performed using enhanced chemiluminescence western blotting substrate (Google Biotechnology, Wuhan, Hubei, China) and detected by ChemiScope3500 Mini System.
4
Metabolic Protein Analysis in Cells
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MPC1 (14462), MPC2 (46141), β-Actin (4970), Akt (9272) were purchased from Cell Signaling Technologies, UCP1 (AB10983), Cytochrome C [7H8.2C12] (AB13575), HMGB1 (AB18256) were purchased from Abcam. 4-hydroxy-tamoxifen (4-OHT) and UK5099 were purchased from Tocris. CL-316,243 (C5796) was purchased from Sigma. U-13C D-Glucose (CLM-1396–5) and U-13C Sodium palmitate (CLM-6059–1) were purchased from Cambridge Isotopes. Sodium palmitate (P9767) was purchased from Sigma Aldrich. DL-[1-14C] 3-hydroxybutyric acid sodium salt (ARC1455) was purchased from American Radiolabeled Chemicals. DL-β-Hydroxybutyric acid sodium salt (H6501) was purchased from Sigma.
5
Cultivation and Characterization of E. cristatum and A. muciniphila
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E. cristatum obtained from the China Center of Industrial Culture Collection (Beijing, China) was cultured according to our previous method [8 (link)]. The stored E. cristatum was activated and cultured on potato dextrose agar (PDA) medium at 28 °C for 72 h. The harvested spores were mixed with distilled water and counted using a hemocytometer. Based on the counting concentration, the working concentration was adjusted to 106 and 108 CFU/mL using distilled water. For the colon-derived A. muciniphila, it was grown in a brain–heart infusion medium under strict anaerobic conditions using a suitable anaerobic chamber (100% N2). The culture was maintained at 37 °C for 48 h, followed by storage at −80 °C for future use. The BCA Protein Assay Kits, total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were purchased from Jiancheng Bioengineering Institute Co. Ltd. (Nanjing, China). The antibodies UCP1 (ab10983, 1:500 dilution), PRDM16 (ab106410, 1:500 dilution), and Cy3-conjugated IgG (ab97075, 1:200 dilution) were purchased from Abcam (Cambridge, MA, USA). Sangon Biotech (Shanghai, China) provided the TRIzol reagent, reverse transcription kit, and SYBR green mixture.
6
Adipocyte Protein Extraction and Western Blot
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Protein was extracted from tissues in RIPA buffer (Boston BioProducts) supplemented with complete protease inhibitor cocktail (Roche). Fat pads were fractionated into adipocytes and non-adipocytes as described [16] (link). Lysates were separated by 4–15% gradient SDS-PAGE and transferred to PVDF membrane (Millipore). The following antibodies were used: Akt (#9272), p-Akt (S473) (#9271), p70S6K (#2708), p-p70S6K (#9205), β-actin (#4970), and α/β-tubulin (#2148), all from Cell Signaling Technology. GR (M-20) was from Santa Cruz. UCP-1 (ab10983), total OXPHOS (ab110143), and Tomm20 (ab56783) were from Abcam. All blots were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
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