Cell lines were grown on coverslips and MBCs were grown on Matrigel coated coverslips overnight. The cells were fixed with 4% paraformaldehyde for 15 min. Cells were analyzed by immunofluorescence according to standard methods. The primary antibody used for immunofluorescence was IGFBP2 (Abcam), N-Cadherin (Cell signaling Technology). The secondary antibody used was Alexa Fluor 594. Nuclei were stained using DAPI. Cell imaging was performed using a Leica DM2500 microscope equipped with a DFC365FX camera and Leica software. For analysis of paraffin embedded and sectioned NeuroD2:Smo/A1mouse MB samples, slides were deparaffinized, dehydrated and antigen retrieval was performed using Tris–EDTA Buffer, pH 9.0 (Abcam). Tissues were blocked with 5% goat serum. The primary antibodies used for IHC were IGFBP2 (#ab188200 Abcam), GFAP (#3670S Cell signaling Technology). Confocal imaging was performed on the Olympus FV1000 at the Emory University Integrated Cellular Imaging Core.
Igfbp2
Manufactured by Abcam
Sourced in United Kingdom, United States
IGFBP2 is a protein that binds to insulin-like growth factors (IGFs) and regulates their biological activities. It is involved in the regulation of cell growth, differentiation, and metabolism.
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Lab products found in correlation
Dfc365 fx camera, by Leica (1 mentions)
Fv1000, by Olympus (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Accublock, by Labnet (1 mentions)
Grp78, by BD (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Fibronectin, by BD (1 mentions)
β actin, by Merck Group (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Mouse elisa kit, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Igfbp 2, by Santa Cruz Biotechnology (1 mentions)
Bh 2 microscope, by Olympus (1 mentions)
Adiponectin, by Abcam (1 mentions)
8 protocols using igfbp2
1
Immunofluorescence and IHC Analysis of IGFBP2 and N-Cadherin in Medulloblastoma
2
Western Blot Analysis of Protein Expression
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30 μg of whole cell lysate, determined using a BCA assay were mixed with an equal volume of 2x Laemmli sample buffer concentrate and 10% 2‐mercaptoethanol (Sigma, S3041‐1VL) and heated at 95°C for 5 min in an AccuBlock™ digital dry bath (Labnet International, D1200). Protein separation was carried out by SDS‐PAGE and then transferred to a nitrocellulose membrane (Bio‐rad, 1620094). Non‐specific binding sites were blocked with 5% milk (Marvel) in tris‐buffered saline TWEEN®20 (TBS‐T) rocking for 60 min at room temperature. Membranes were incubated with specific primary antibodies diluted in fresh blocking solution overnight at 4°C: α‐tubulin (Millipore, 05‐829; 1:5000), IGFBP‐2 (Abcam, 109284; 1:1000), GRP78 (BD Transduction Laboratories™, 610979). Membranes were washed with TBS‐T and incubated with secondary antibodies, (Sigma, A0545 or A4416; 1:2000). Proteins were visualised exposing with the ChemiDoc MP Imaging System with Image Lab software (Bio‐Rad) once treated with Clarity Western ECL Substrate (Bio‐Rad, 1705061). Densiometric analysis of protein bands was carried out using Image J (NIH).
3
Western Blot Analysis of Cell Signaling Proteins
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Western blot analysis was performed by using standard protocols. Cell lysate was extracted using RIPA buffer (Cell Signaling) supplemented with 1mM phenylmethylsulfonyl fluoride (PMSF) just before use. Primary antibodies used were: IGFBP2 (ab13649, abcam), fibronectin (610077, BD), CD44 (ab157107, abcam), vimentin (5741, Cell Signaling), and β-actin (A5316, Sigma Aldrich); the goat-anti-mouse (SA00001-1) or rabbit (SA00001-2) second antibodies were bought from Proteinteck Company.
4
Quantification of IGFBP Proteins in Hepatocytes
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IGFBP2 protein abundance was examined in hepatocyte cell lysates by Western blotting [21 (link)] using IGFBP2 (Santa Cruz Biotechnology, Heidelberg, Germany) and GAPDH (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. IGFBP2, IGFBP3, and IGF1 content was measured in unprocessed culture supernatants obtained from 24 h serum-free hepatocyte cultures, as well as in blood plasma using specific mouse Elisa kits (Abcam, Cambridge, UK/R&D systems, Wiesbaden, Germany).
5
Multimodal Immunohistochemical Profiling of Bone, Cell, and Cytospin Samples
6
Western Blot Analysis of Piezo1 and Related Proteins
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Western blot was performed as in our previous work [18 (link)] with a little modification. Briefly, 100 μg protein extracted from HCC cells was loaded and resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) for Piezo1 detection, and 20 μg protein extract for other target protein detection. The conditions of membrane transfer were optimized as 350 mA, 210 min for Piezo1, 300 mA, 45 min for von Hippel‐Lindau (VHL), 300 mA, and 90 min for other target proteins. The diluted primary antibodies were as follows: integrin β1 (1:1000, Cell Signal Technology, Boston, MA, USA), Piezo1 (1:500, Abcam), COL1 (1:1000, Affinity), α‐tubulin (1:5000, Proteintech), HIF‐1α (1:1000, Abcam), VHL (1:1000, Abcam), IGFBP2 (1:1000, Abcam), CXCL16 (1:1000, Abcam), L‐vascular endothelial growth factor A (L‐VEGFA) (1:1000, Proteintech), ubiquitin (UB) (1:1000, Cell Signal Technology). The secondary horseradish peroxidase (HRP)‐conjugated antibody (Proteintech) was diluted at 1:5000.
7
Western Blot Protein Quantification
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Tissues and cells were homogenized then lysed in RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitors. 15–30 µg of each sample was denatured and separated in 10% and 12% polyacrylamide gels, then transferred to immobilon-P membranes (Millipore) [30 (link)]. Because many of the proteins are of similar molecular weight, parallel gels were run and transferred before cutting membranes for antibody hydridization; equal amounts of protein were loaded in each lane. For quantification purposes, chemiluminescent signals were normalized to that of GAPDH /β-tubulin on the same blot. The following antibodies were used (IGFBP2 #ab188200 abcam, IGFBP2 #3922S Cell signaling Technology, β-tubulin #SC53140 Santa Cruz Biotechnology, Lamin B1 #13435S Cell signaling Technology, N-Cadherin #13116 Cell signaling Technology, Slug #9585 Cell signaling Technology, E-Cadherin #3195 Cell signaling Technology, MMP-3 #14351 Cell signaling Technology, MMP-7 #71031 Cell signaling Technology, MT1-MMP #13130 Cell signaling Technology, MMP-9 #13667 Cell signaling Technology, STAT3 #12640S Cell signaling Technology, Phospho- STAT3 (Tyr705) #9145 Cell signaling Technology).
8
Resveratrol Nanoparticles for Renal Protection
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Resveratrol (C14H12O3; > 98% pure) was purchased from Shaanxi Sciphar Biotechnology Co., Ltd. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine and polyethylene glycol were purchased from Sigma Inc. Res-NPs were constructed as described in our previous study [12 (link)]. Phosphate-buffered saline (PBS), saline, apoptosis detection kit (Annexin VFITC/PI), protein extraction kit, malondialdehyde (MDA) kit, glutathione peroxidase (GSH-Px) kit, terminal deoxynucleotidyl transferase-mediated deoxyuridine-triphosphate nick end-labeling (TUNEL) apoptotic detection kit, and primary antibodies (BAX, caspase3, and BCL-2) were all purchased from Nanjing Key Gen Biotech. Co., Ltd. Blood urea nitrogen (BUN), C-reactive protein (CRP), cystatin C protease inhibitors (Cys-C), and serum creatinine (SCr) were all obtained from Weiteman, Co., Ltd., (Nanjing, China).
The following instruments were used: automatic biochemical analyzer (Hitachi 7100, Japan), microplate reader (ELx800, BioTek, USA), flow cytometer (FACSCalibur, Becton-Dickinson, USA), inverted fluorescence microscope (IX51, Olympus, Japan), Western electrophoresis apparatus (164-5051, Bio-Rad, USA), spectrophotometer (UV-2540, Shimadzu, Japan), and gel imager (Gel Doc XR, Bio-Rad, USA). Primary antibodies (IGFBP2, AAab136494; KIM-1, ab47634; NGAL, and ab63929) were purchased from Abcam (Cambridge, UK).
The following instruments were used: automatic biochemical analyzer (Hitachi 7100, Japan), microplate reader (ELx800, BioTek, USA), flow cytometer (FACSCalibur, Becton-Dickinson, USA), inverted fluorescence microscope (IX51, Olympus, Japan), Western electrophoresis apparatus (164-5051, Bio-Rad, USA), spectrophotometer (UV-2540, Shimadzu, Japan), and gel imager (Gel Doc XR, Bio-Rad, USA). Primary antibodies (IGFBP2, AAab136494; KIM-1, ab47634; NGAL, and ab63929) were purchased from Abcam (Cambridge, UK).
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