Cells were lysed on ice in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Beyotime, Haimen, China). Total and nuclear proteins were extracted according to the manufacturer’s instructions (Beyotime, Haimen, China). Protein samples (50 μg) were separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked in TBST containing 5% fat-free milk at room temperature for 1 h. After washing with TBST, the membranes were probed with primary antibodies against C/EBPα, FABP4, PPARγ, RhoA, ROCK1, ROCK2, ERK1/2, β-actin, PCNA (all Proteintech, Wuhan, China), and p-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). The membranes were washed in TBST and incubated with secondary antibody (Proteintech, Wuhan, China). The signals were developed by ECL reagents (Beyotime, Haimen, China).
Fabp4
Manufactured by Proteintech
Sourced in China
FABP4 is a protein that binds and transports fatty acids. It plays a role in lipid metabolism and adipocyte differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Pparγ, by Proteintech (3 mentions)
C ebpα, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Rock1, by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Rock2, by Proteintech (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Erk1 2, by Proteintech (1 mentions)
Goat anti mouse igg, by Wuhan Servicebio Technology (1 mentions)
Goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Srebp1, by Proteintech (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Goat pab to rb igg, by Abcam (1 mentions)
Pdgfrα, by Abcam (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Skimmed milk powder, by Merck Group (1 mentions)
8 protocols using fabp4
1
Western Blot Analysis of Adipogenic Markers
2
Protein Expression Profiling in PSCs and Preadipocytes
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In PSCs, the expression levels of MYOG, MYOD, MHC and CXXC5 protein were detected. The protein expression levels of FABP4, PPARγ, SREBP1 and FASN were detected in preadipocytes. Transfected cells were lysed in RIPA buffer with 1% PMSF. 5×Buffer was added to the sample and denatured at 100 °C for 10 min. SDS-PAGE gel electrophoresis was performed at 80 V 30 min, 120 V 90 min. Then transferred them onto a PVDF membrane and non-specific binding was blocked with 5% non-fat milk in PBS for 1 h. Then, they were incubated with 1:1000 diluted polyclonal rabbit MYOG (Abclonal, China), MYOD (Proteintech, China), CXXC5 (Bioss, China), 1:500 diluted polyclonal mouse MHC (DSHB, America), 1:1000 diluted polyclonal rabbit FABP4 (Proteintech, China), PPARγ (Proteintech, China), SREBP1 (Proteintech, China) and FASN (Proteintech, China) at 4 ℃ overnight. The blots were subsequently incubated with secondary antibody (1:10000) for 1 h. Secondary antibody include goat anti-mouse IgG (Servicebio, China) and goat anti-rabbit IgG (Servicebio, China). GAPDH (Servicebio, China) was used as an endogenous protein for normalization. Image J software was used to conduct quantitative analysis of western blot results according to the gray value of the strip.
3
Immunofluorescence Analysis of Thoracic Aortic PVAT
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Sections of the thoracic aortic PVAT were prepared and incubated with a primary antibody mixture of UCP1 (dilution: 1:100, Proteintech, Wuhan, China), FABP4 (dilution: 1:100, Proteintech, Wuhan, China), PDGFRα (dilution: 1: 100, Abcam, Cambridge, UK), or Ki67 (dilution: 1:100, Abcam, Cambridge, UK) at 4°C overnight. The samples were protected from light exposure, and Goat pAb to Rb IgG (dilution: 1:500, Abcam, Cambridge, UK) secondary antibody mixture was added and incubated for 1 h at room temperature before sealing with a DPAI-containing sealer (Beyotime, Shanghai, China). Finally, the stained tissues were examined under a fluorescence microscope (Leica, Weztlar, Germany). The fluorescence index was determined using Image J software (National Institutes of Health, USA).
4
Western Blot Protein Expression Analysis
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Cells were extracted using a protein lysis buffer supplemented with protease inhibitor cocktail (Boster, China). Protein concentration was determined using a BCA kit (Boster, China). Then, samples were loaded at 20–30 μg/lane and separated on a 10% precast SDS–PAGE gel, followed by transfer onto PVDF membrane (Millipore, USA). After blocking with 5% skimmed milk powder (Sigma–Aldrich, USA) for 3 h, the membrane was incubated overnight at 4 °C with the appropriate primary antibody: against PPARγ (A0270, Abclonal, China), β-actin (AC038), FABP4 (A0232), CEBP-α (A0904), MYOZ2 (A6468), or AHCY (10,757, Proteintech, China). Then, IRDye® 800CW rabbit secondary antibodies (926-32,211, LI-COR, USA) were used to detect the primary antibodies, and the membrane was imaged using a far-infrared light scanning system (LI-COR, USA).
5
Quantitative Western Blot Analysis of PVAT Proteins
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Total proteins were extracted from rat thoracic aorta PVAT using RIPA lysis buffer supplemented with a protease and phosphatase inhibitor mixture (Thermo Fisher Scientific, Waltham, USA). The total protein content was measured using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). Protein (30 μg) was separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels ranging from 10 to 12%. The separated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, CA, USA) and were then blocked with 5% skimmed milk (Bio-Rad, CA, USA) for 1 h. Following this, the membranes were washed three times with TBST buffer and incubated overnight at 4°C with respective primary antibodies, such as BMP4, UCP1, FABP4, PGC1α, GAPDH (Proteintech, Wuhan, China), P-p38/MAPK, and p38/MAPK (Cell Signaling Technology, MA, USA). After TBST washing, membranes were probed with secondary antibodies (goat anti-mouse from Proteintech and goat anti-rabbit from Cell Signaling Technology) for 2 h at room temperature. Following three additional TBST washes, we applied an enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, USA), and the membranes were visualized using a chemiluminescence imaging system (Bio-Rad, CA, USA). The intensity of the bands was quantified using ImageJ software (National Institutes of Health, USA).
6
Western Blot Analysis of Adipocyte Markers
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Cells were collected, washed with PBS and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer with 2% protease inhibitor for 30 min. After centrifugation at 12,000 × g for 20 min, the isolated proteins were subjected to 10% or 15% SDS-PAGE and transferred to the PVDF membranes. After blocking in 5% dehydrated milk for 1–3 h, membranes were then incubated with primary antibodies against PPARγ (Abcam, Cambridge, MA, USA; 178,860), C/EBPα (Proteintech, Rosemont, IL, USA; 18,311–1-AP), fatty acid binding protein 4 (FABP4) (Proteintech; 12,802–1-AP), CDC20 (Abcam; ab183479), and β-catenin (Proteintech; 51,067–2-AP) overnight. Membranes were washed with PBS, incubated with the secondary antibody solution for 1 h, and then washed with PBS for three times. The immunoreactive protein bands were observed using an enhanced chemiluminescence (ECL) kit (CWBIO, Beijing, China). The protein levels were quantified through ImageJ software (National Institutes of Health, Bethesda, Maryland). GAPDH was used as internal control.
7
Western Blot Analysis of Protein Targets
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Western blotting was performed as described in our previously published study [56 (link)]. The total protein of cells was extracted and separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes with a transfer apparatus (Bio-Rad, Hercules, CA, USA). After block, primary antibodies, including EGR3, PPARγ, HDAC6, FABP4, CIDEC, and GAPDH (Proteintech, Wuhan, China) were treated overnight at 4 °C, and second antibody was used for the next ECL (Advansta, Menlo Park, CA, USA) detected.
8
Histological Analysis of ingWAT
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Collected ingWATs were fixed in 10 % neutral buffered formalin for >24 h. The tissues were then embedded in paraffin, sectioned at 4-5 μm, and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis in ingWAT sections was performed using primary antibodies of fatty acid binding protein 4 (FABP4) (Proteintech, Wuhan, China), KI67 (Proteintech), and uncoupling protein 1 (UCP1) (Proteintech) and visualized with 3, 3′-diaminobenzidine (DAB) staining (ZSGB-Bio, Beijing, China). The slides were examined under a digitalized microscope camera (Olympus, Tokyo, Japan) and further analyzed using the ImageJ software (NIH, Bethesda, MD).
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