The liver tissues were isolated and pestled, and were lysed in radioimmunoprecipitation assay buffer. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to separate equal amounts of cell lysates, and were transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA). After incubation with the primary antibodies overnight at 4°C, and corresponding secondary antibodies for 2 hours at room temperature. Finaly, dropped electrochemiluminescence liquid for 1 minute, signals were detected using a chemiluminescence instrument (Imagequant LAS500, Cytiva, USA). Primary antibodies were as follows: CMV-IE1 and IE2 (1:500, cat. ab53495; Abcam, Cambridge, UK), P27 (1:1000, cat. A16722; ABclonal, Wuhan, China), P21 (1:1000, cat. bs-55160R; Bioss, Beijing, China), CEBPα (1:1000, cat. A0904; ABclonal, Wuhan, China), HNF4α (1:1000, cat. A13998; ABclonal, Wuhan, China), and β-actin (1:2000; Santa Cruz, CA). The secondary antibodies used were horseradish peroxidase–conjugated sheep anti-mouse IgG (1:500; Absin, Shanghai, China) and anti-rabbit (1:10,000; Absin, Shanghai, China).
C ebpα
Manufactured by Bioss Antibodies
Sourced in China
C/EBPα is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. It plays a key role in the regulation of gene expression involved in cellular processes such as differentiation, proliferation, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Bioss Antibodies (2 mentions)
Adiponectin, by Bioss Antibodies (2 mentions)
Pparγ, by Bioss Antibodies (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Cmv ie1 and ie2, by Abcam (1 mentions)
Hnf4α, by ABclonal (1 mentions)
Horseradish peroxidase conjugated sheep anti mouse igg, by Absin (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 500, by Cytiva (1 mentions)
Flag tag (flag), by Beyotime (1 mentions)
Pgc 1α, by ZenBio (1 mentions)
P smad1 5 8, by Affinity Biosciences (1 mentions)
Ha tag, by Beyotime (1 mentions)
P acc, by Sangon (1 mentions)
Goat anti rabbit igg hrp secondary antibody, by CWBIO (1 mentions)
P ikkα β, by Bioss Antibodies (1 mentions)
P p38 mapk, by Bioss Antibodies (1 mentions)
Pparγ, by Affinity Biosciences (1 mentions)
P38 mapk, by Bioss Antibodies (1 mentions)
P erk1 2, by Bioss Antibodies (1 mentions)
5 protocols using c ebpα
1
Liver Protein Expression Analysis
2
Immunoblotting for Adipogenesis Regulators
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Antibodies from Affinity Biosciences: p-Smad1/5/8 (1:1000, #AF8313), Smad1/5/8 (1:1000, #AF0614), PPARγ (1:1000, #bs-4590R), UCP1 (1:1000, #72298). Antibodies from Beyotime: HA tag (1:1000, #AH158), Flag tag (1:1000, #AF519). Antibodies from Sangon Biotech: p-ACC (1:1000, #D155180), ACC (1:1000, #D155300). Antibodies from Abcam: BMP8A (1:1000, #154373). Antibodies from ZenBio: p-AMPK (1:1000, #R26252), AMPK (1:1000, #380431), PGC-1α (1:1000, #381615). Antibodies from CWBIO: goat anti-Rabbit IgG HRP secondary antibody (1:4000, #CW0103S), goat anti-Mouse IgG HRP Conjugated (1:4000, # CW0102). Antibodies from Bioss: p38 MAPK (1:1000, #bs-0637R), p-p38 MAPK (1:1000, #bs-2210R), β-actin (1:2000, #bs-0061R), p-JNK (1:1000, #bs-1640R), JNK (1:1000, #bs-2592R), C/EBPα (1:1000, #AF6333), p-Smad2/3 (1:1000, #AF3367), Smad2/3 (1:1000, #AF6367), p-ERK1/2 (1:1000, #AF1015), ERK1/2 (1:1000, #AF0155), p-p65 (1:1000, #AF2006), p65 (1:1000, #AF5006), p-IKKα/β (1:1000, #AF3013), IKKα/β (1:1000, # AF6014).
3
Adipose Tissue and Capillary Formation
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The excised neo-tissue samples from the silicone tubes were fixed in 10% formalin, dehydrated with a graded alcohol series, and embedded in paraffin. Sections with a thickness of 4 μm were stained with hematoxylin and eosin (H&E). Images of the histologic sections were examined microscopically (Leica, Germany) at a magnification of ×100. To quantitatively analyze the area of intact adipocytes and the number of mature capillaries, images in 8 random fields per section from each group (×100 magnification) were examined by using ImageJ software. Immunochemical staining for C/EBP-α (Bioss, China, cat.bs1630R), PPARγ (Bioss, China, cat.bs4888R), Adiponectin (Bioss, China, cat.bs0471R), and CD31 (Sino Biological, China, cat.50408-T16) was performed to determine the extent of adipose tissue and blood vessel formation in new growth tissues at the 4th week.
4
Western Blot Analysis of Adipogenic Markers
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The protein from cells were extracted and quantified by BCA Kit (Beyotime Biotechnology, P0012S) according to the manufacturer’s instruction. The samples were subjected to SDS-PAGE and subsequently transferred to PVDF membranes. Antibodies against PPARγ (1:1000, Bioss, bs-0530 R), C/EBPα (1:1000, Bioss, bs-24,540 R), IGF1 (1:1000, Bioss, bs-0014 R), PPARGC1B (1:1000, Bioss, bs-7534 R) or β-actin (1:5000, Bioss, bsm-33,036 M) were incubated with the membranes at 4°C overnight with constantly shaking. Then the membranes were treated with an HRP-conjugated secondary antibody. The signal was visualized with an enhanced chemiluminescence substrate.
5
Histological Analysis of Adipose Tissue
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The excised neo-tissue samples from the silicone tubes were xed in 10% formalin, dehydrated with a graded alcohol series, embedded in para n. Sections with a thickness of 4 µm were stained with hematoxylin and eosin (H&E). Images of the histologic sections were examined microscopically (Leica, Germany) at magni cation of 100×. To quantitatively analyze the area of intact adipocytes and the number of mature capillaries, images in 8 random elds per section from each group (100× magni cation) were examined by using ImageJ software. Immunochemical staining for C/EBP-α (Bioss, China, cat.bs1630R), PPARγ (Bioss, China, cat.bs4888R), Adiponectin (Bioss, China, cat.bs0471R) and CD31 (Sino Biological, China, cat.50408-T16) was performed to determine the extent of adipose tissue and blood vessel formation in new growth tissues on the 4th week.
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