Cells (1 × 107) were disrupted with IP lysis buffer containing protease inhibitor cocktail (Bimake). Protein aliquots (1000 μg) were incubated with 20 μL of Dynabeads Protein A (Invitrogen) for 1 hour at 4°C for pre‐clearing. The samples were incubated with 2 μg anti–CEBPB (ab32358; Abcam) or 2 μg IgG overnight at 4°C with mild shaking. IgG was used as a negative control. Then 20 μL of Dynabeads Protein A was added to samples and incubated for 2 hours at 4°C. The beads were washed 3 times with cold lysis buffer, then resuspended in 20 μL of 1× loading buffer diluted with lysis buffer and boiled for 5 minutes. The samples were analyzed by western blotting. The antibodies used for western blot detection were anti–PGC1α (ST‐1202; Millipore) and anti–CEBPB (ab32358; Abcam).
Ab32358
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab32358 is a laboratory equipment product offered by Abcam. It serves a core function related to laboratory analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further information about the intended use or specific capabilities of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab92547, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab76003, by Abcam (2 mentions)
Ab272718, by Abcam (2 mentions)
Ab6528, by Abcam (2 mentions)
Ab134146, by Abcam (2 mentions)
Ab51608, by Abcam (2 mentions)
Ab54365, by Abcam (2 mentions)
Ab76417, by Abcam (2 mentions)
Ab6258, by Abcam (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Hrp conjugated goat anti rabbit igg h l, by Abcam (2 mentions)
Ab32572, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sc 377154, by Santa Cruz Biotechnology (1 mentions)
Ab209350, by Abcam (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Cy5561, by Abways (1 mentions)
Ab92536, by Abcam (1 mentions)
29 protocols using ab32358
1
Immunoprecipitation of CEBPB and PGC1α
2
High-Fat Diet and Adipogenesis Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HFD (TP23400, 60% high-fat model) was ordered from Nantong Trophy Feed (Nantong, Jiangsu, China). Sophoridine (HPLC, ≥98.6%, CAS: 6882-68-4) was purchased from Chengdu Durst (Chengdu, China). Antibodies against peroxisome proliferator-activated receptor gamma (PPARγ, ab209350) and CCAAT enhancer binding protein alpha (C/EBPα, ab32358) were purchased from Abcam. Antibodies against adipose triglyceride lipase (ATGL, 2138) and glucose transporter 4 (GLUT4, 2213) were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against fatty acid binding protein 4 (FABP4, sc-271529), cyclinB (sc-166152), P21 (sc-6246), cyclin-dependent kinase 4 (CDK4, sc-23896), cAMP-response element binding protein (sc-377154), and β-actin (sc-47778) were obtained from Santa Cruz (Dallas, TX, USA). Antibodies against CCAAT enhancer binding protein beta (CEBPβ, CY5065), cyclinD1 (CY5404), cyclinE2 (CY5821), c-Src (CY5459), vascular endothelial growth factor (VEGF, CY10042), vascular endothelial growth factor receptor 2 (VEGFR2, CY5385), phosphoinositide-3 kinase P85 alpha (P85α-PI3K, CY5355), AKT1/2/3 (CY5561), ERK1/2 (CY5487), P-P85α-PI3K (CY6427), P-AKT1/2 (Tyr315/316/312, AY0423), and P-ERK1/2 (Tyr185/Y187, CY5044) were purchased from Abways (Shanghai, China).
3
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were lysed with RIPA containing 1% protease and phosphate inhibitor cocktail (P8340; Sigma-Aldrich). Proteins were run on 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies at 4°C overnight and probed by secondary antibodies. Blots were visualized with enhanced chemiluminescence (WBKLS0500; Millipore, Temecula, CA) and the ChemiDoc Touch detection system (Bio-Rad). The following primary antibodies were used: slug (9585; Cell Signaling Technology, Danvers, MA; 1:1000), β-catenin (8480; Cell Signaling Technology; 1:1000), p-eIF2α (3398; Cell Signaling Technology; 1:1000), p-GSK3β (5558; Cell Signaling Technology; 1:1000), GAPDH (ab8245; Abcam, Cambridge, England; 1:1000), C/EBPβ (ab32358; Abcam; 1:1000), N-cadherin (ab76057; Abcam; 1:1000), MMP-2 (ab92536; Abcam; 1:1000), AKT (ab8805; Abcam; 1:1000), p-AKT (ab81283; Abcam; 1:1000), vimentin (ab92547; Abcam; 1:5000), and cleaved caspase-3 (19677-1-AP; Proteintech, Rosemont, IL; 1:500).
4
Adipocyte Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mature adipocytes were harvested in cold RIPA lysis buffer (Beyotime, China) containing 1% phenylmethylsulfonyl fluoride (PMSF, Beyotime).The concentrations of protein were determined by using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were loaded and analyzed by10% SDS-PAGE and transferred to 0.2-μm PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were probed with the following primary antibodies overnight at 4 °C: rabbit polyclonal β-actin (Biosharp, China, BL005B, diluted 1:1000), monoclonal rabbit PPAR-γ (Abcam, ab178860, diluted 1:1000), polyclonal rabbit C/EBP-β (Abcam, ab32358, diluted 1:1000), and C/EBP-α (Abcam, ab40761, diluted 1:1000). The membranes were washed 3 times with TBST and incubated with peroxidase-conjugated goat anti-rabbit IgG (Biosharp, China, BL003A, diluted 1:5000) for 1 h at room temperature. All bands were visualized by using Beyo ECL Plus (Beyotime, China, P0018).
5
ChIP Assay for CEBPB and CEBPD
6
Western Blot Analysis of Protein Biomarkers
7
Co-Immunoprecipitation of YAP, TEAD4 and C/EBPβ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-IP was conducted according to the instructions of the Pierce™ Classic Magnetic IP/Co-IP Kit (#88804, Thermo Fisher Scientific). In brief, cell lysates were incubated with anti-YAP antibody (#ab52771, Abcam), anti-C/EBPβ (#PA5-27244, Thermo Scientific) antibody or rabbit IgG overnight at 4°C to form antigen/antibody complexes. These antigen/antibody complexes were incubated with protein A/G magnetic beads at room temperature for 1h. Magnetic beads were then collected using a magnetic scaffold and washed twice with IP lysis/washing buffer and once with PBS. Magnetic bead precipitates were collected by centrifugation and 2×loading buffer was added. These magnetic beads-antigen-antibody mixtures were then boiled at 100°C for 10min. The levels of YAP, TEAD4, C/EBPβ and acetylated lysine were detected by western blot using anti-YAP (#ab56701, Abcam), anti-TEAD4 (#sc-101184, Santa Cruz), anti-C/EBPβ (#ab32358, Abcam) and anti-acetyl lysine (#ab21623, Abcam) antibodies.
8
Adiponectin Signaling and Autophagy Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against phospho-AMPK (2531) at Thr172, AMPK (2603), phospho-IKKα (Ser176)/IKKβ (Ser177; 2078S), IKKα (2682), IKKβ (2370), phospho-IκB at Ser32/36 (9246), IκB (4814), phospho-ULK1 at Ser317 (6887), and ULK1 (8054) were from Cell Signaling. Anti-PGC1α (ST1204) was purchased from Millipore. Antibodies against AdipoR1 (ab126611), AdipoR2 (ab77612), UCP1 (ab23841), and C/EBPβ (ab32358) were from Abcam. Anti-LC3B (L7543) from Sigma was used for Western blot, analysis and Anti-LC3 pAb (PM036) from MBL International was used for staining. Anti-adiponectin and anti-β-tubulin were kindly provided by Dr. Lily Dong (University of Texas Health Science Center at San Antonio, San Antonio, TX) as described previously (Alsted et al., 2009 (link); Meng et al., 2017 (link)). AdipoRon (924416–43-3) and 5Z (253863–19-3) were obtained from Cayman Chemical Company. IRAK 1/4 inhibitor I (I5409) and rapamycin (R8781) were from Sigma-Aldrich. 3-MA (189490) was purchased from EMD Millipore. Two recombinant mouse adiponectin (full-length and mutated C39A) proteins (ab62957 and ab94676) were purchased from Abcam. Mouse ST2/IL-1 R4 antibody (MAB10041) was from R&D Systems.
9
Immunofluorescent Analysis of PRMT1 and C/EBPβ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After DMI induction for 1 day, 3T3-L1 cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% of Triton X-100 in PBS for 20 min. Samples were then blocked in 4% bovine serum albumin in PBS for 1 h at RT and incubated with mouse anti-PRMT1 antibody (sc-166963, Santa Cruz) and rabbit anti-C/EBPβ antibody (ab32358, abcam) in 1% bovine serum albumin/PBS overnight at 4 °C. Washed three times with PBS, cells were then incubated with antimouse IgG secondary antibody–conjugated Alexa Fluor 488 and anti-rabbit IgG secondary antibody–conjugated Alexa Fluor 555 for 1 h at RT. Nuclei were stained by DAPI, and samples were analyzed by confocal microscopy (Leica).
10
ChIP-qPCR for C/EBPβ and HAS2-AS1 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect C/EBPβ and HAS2-AS1-binding sites, ChIP Assay Kit (ABCAM) was used to perform chromatin immunoprecipitation (ChIP) according to the instructions and established protocol (Ke et al., 2020 (link)); 200–1,000 bp DNA fragments were obtained by sonication. HFL-1 cells were cross-linked with formaldehyde. Subsequently, non-specific IgG antibodies (ab172730, Abcam) and C/EBPβ antibody (ab32358, Abcam) were precipitated with chromatin overnight at 4°C. NaCl was used to reverse DNA cross-linking, qRT-PCR was used to detect HAS2-AS1 level, and 2–ΔΔCT was used to calculate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!