Protein lysates were prepared with mCherry expressed tumors from nf1a+/−; nf1b−/−; p53m/m fish. Briefly, mCherry-positive tumor mass were harvested from tricaine-anesthetized tumor fish, and were lysed on ice in 1 × RIPA (Cell Signaling, Danvers, MA, USA) containing, PhosSTOP phosphatase inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and complete protease inhibitor tablet (Roche). The inhibitors were prepared following the manufacturer's recommendation. Protein lysates were separated by gel electrophoresis, transferred to PVDF membranes and probed overnight at 4 °C with the following primary antibodies: anti-PDGFRA (Cell Signaling; 5241; 1:500), anti-p-tyrosine (EMD Millipore, Billerica, MA, USA; 05-321; 1:1000), anti-β-tubulin (Cell Signaling 2146; 1:2000), anti-AKT (Cell Signaling; 9272; 1:1000), anti-p-AKT (Cell Signaling; 4060; 1:1000), anti-p-ERK1/2 (Cell Signaling; 4377;1:1000) and anti-ERK1/2 (Cell Signaling; 9102; 1:1000). Primary antibody binding was visualized on X-ray film using anti-mouse-HRP (Cell Signaling; 7076; 1:10 000) or anti-rabbit-HRP (Cell Signaling; 7074; 1:10 000) secondary antibodies along with SuperSignal West Dura or Femto (Thermo Fisher Scientific, Waltham, MA, USA) chemiluminescent substrates. Stripping was performed using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) according to the manufacturer's protocol.
Anti pdgfra
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PDGFRA is a primary antibody that targets the platelet-derived growth factor receptor alpha (PDGFRA) protein. PDGFRA is a receptor tyrosine kinase that plays a role in cell growth, proliferation, and survival. This antibody can be used to detect and study the expression and localization of PDGFRA in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfap, by Cell Signaling Technology (2 mentions)
Anti notch1, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Phosstop phosphatase inhibitor cocktail tablet, by Roche (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti nestin, by Santa Cruz Biotechnology (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti pdgfra
1
Protein Lysate Preparation from Zebrafish Tumors
2
Immunoblotting Technique for Protein Analysis
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Immunoblotting was performed as described in our previous reports [31 (link), 41 (link), 56 (link), 58 (link)]. In brief, cells were lysed and total protein was quantified using the Bradford assay (Bio-Rad Laboratories Inc.). An equal amount of total protein (20–25 μg) in each sample was loaded onto an SDS-PAGE gel. After transferring to PVDF membrane, the blot was incubated with antibodies. Antibodies were diluted as follows: anti-Nestin (Santa Cruz Biotechnology, Inc., 1:200), anti-NOTCH1 (Cell Signaling Technology, 1:1000), anti-GFAP (Cell Signaling Technology, 1:1000), anti-PDGFRA (Cell Signaling Technology, 1:1000), anti-B-Actin (SigmaAldrich Co. LLC, 1:10000), and anti-GAPDH (Santa Cruz Biotechnology, Inc., 1:1000). Images were taken using a ChemiDoc MP System (Bio-Rad Laboratories Inc.).
3
Western Blot Analysis of Protein Expression
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Cells were treated with tRNA/miR-34a or tRNA/MSA (5 nM, 48 hours) or vehicle, and were collected in ice-cold PBS and centrifuged. Whole-protein extracts were obtained by lysing cell pellets with RIPA Lysis Buffer [Tris-HCl 50 mM, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and supplemented with cOmplete Protease Inhibitor Cocktail (Roche)]. Protein concentrations were determined by bicinchoninic acid-based assay (Pierce BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA). Whole-protein extracts (25 μg) were resolved by Tris-Acetate (7%) or Tris-Glycine (10%) and transferred to polyvinylidene difluoride membranes using a Mini Gel/Blot System (Invitrogen). After blocking with 5% non-fat milk or bovine serum albumin, membranes were incubated with primary antibodies: anti-PDGFRa (#3174), anti-Notch1 (#3608) (Cell Signaling, Boston, MA) or anti-ß-actin (MA1-91399, ThermoFisher Scientific); and horseradish peroxidase-conjugated secondary anti-mouse (#32430) or anti-rabbit (#32260) antibodies (Invitrogen). The chemiluminescence signal was detected using Luminata Forte Western HRP substrate (Millipore, Billerica, MA), SuperSignal West Pico or SuperSignal West Femto (both from Thermo Scientific) and was visualized with a digital image analyzer (FluorChem E, ProteinSimple, San Jose, CA).
4
Western Blot Analysis of Cell Signaling
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Cell lysates were prepared as previously described [11 (link)]. Whole cell extract of 30 μg protein was resolved in SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk/PBS for 30 min and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study include anti-PDGFRA (Cell Signaling, 3174T), anti-COL1A1 (Proteintech, 67288-1-Ig), anti-ACTA2 (α-SMA, ABclonal, A7248), anti-SM22 (TAGLN, Proteintech, 10493-1-AP ), anti-PROX1 (Boster, BA2390), anti-PDPN (Proteintech, 11629-1-AP), anti-VEGF-A (Immunoway, YT5108), anti-CD31 (Proteintech, 11265-1-AP), anti-VEGFR2 (Proteintech, 26415-1-AP), anti-VCAM (Proteintech, 11444-1-AP), anti-VEGFR3 (ABclonal, A5605), anti-ETAR (EDNRA, Santa Cruz, sc-135902), anti-ICAM (Proteintech, 10831-1-AP), and anti-β-actin(Sigma, A5441). Anti-IR Dye 800 or Dye 680 anti-rabbit or anti-mouse IgG antibodies (LI-COR Biosciences) were used as the secondary antibodies. An Odyssey system (LI-COR Biosciences) was used for the detection of proteins of interest.
5
Histological and Immunohistochemical Analysis of Formalin-Fixed Mouse Brains
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Formalin-fixed brains (10% formalin for 24 h) were serially sectioned in the coronal plane and processed in paraffin by the Northwestern Mouse Histology and Phenotyping Laboratory. Sections cut at 5-µm were used for histologic and immunohistochemical staining. Hematoxylin and Eosin (H&E) staining were performed using standard protocols. IHC was performed with an automated IHC system (Ventana Medical Systems) with the following antibodies: anti-ATRX (abcam #ab188027, 1:100), anti-GFAP (Cell Signaling Technology #3670S, 1:50), anti-Ki67 (Cell Signaling Technology #12202, 1:400), anti-EMA (Roche Diagnostics, # 05878900001), and anti-Olig2 (Millipore #AB9610, 1:500). IHC was performed using a Vectastain Elite kit (Vector Laboratories #AK-5001) as described previously with the following antibodies: anti-PDGFRA (Cell Signaling Technology, #3174T, 1:1000), anti-pERK1/2 (ABclonal, #AP0472, 1:100), anti-Iba1/AIF-1 (Cell Signaling Technology, #17198T, 1:1500), and anti-H3.3G34R (abcam, #ab254402, 1:1000).23 (link) Rabbit and mouse antibodies were diluted in 2% BSA solution.
6
Overexpression and Knockdown of Key Genes
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Adv-vectors (KLF4, PDGFRA and NAMPT) were used to overexpress target genes, and sh-RNAs (KLF4, PDGFRA and NAMPT) were used to knock down target genes. All were purchased from Viogene Bio. (Jinan, Shandong, China). The KLF4 luciferase reporter plasmid (pGL4-KLF4) was purchased from Yeasen Bio. (Shanghai, China). A fluorescent dual luciferase reporting system and murine leukemia virus reverse transcriptase were obtained from Promega Co. (Mannheim, Germany). The SA-β-gal-kit was obtained from Beyotime (Haimen, China). The MitoSOX Mitochondrial Superoxide Indicator was obtained from Yeasen (Shanghai, China). The anti-p21 antibody was obtained from HUABIO (Hangzhou, China). The anti-PDGF-BB, anti-PDGFRA, anti-PDGFRB, anti-KLF4, anti-NAMPT, anti-PAI-2, anti-uPA and anti-β-actin antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Other reagents were purchased from Sigma (St. Louis, MO, USA).
7
Western Blot Analysis of Fibroblast Proteins
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NHLFs were lysed in buffer (6.1 mmol/L Na 2 HPO 4 , 4.5 mmol/L NaH 2 PO 4 , 88.4 mmol/L NaCl, 36.6 mmol/L LiCl, 36.6 mmol/L SDS, 24.1 mmol/L sodium deoxycholate, and 1% Triton X-100) and complete protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation, and supernatants were denatured at 100 C in Tris-glycine SDS sample buffer. Protein samples (10 mg) were subjected to electrophoresis on a 10% reducing SDS-PAGE gel (Bio-Rad) and blotted onto a nitrocellulose or polyvinylidene difluoride membrane (Bio-Rad Laboratories Incorporated). Membranes were blocked for 2 hours to overnight in Tris-buffered saline, 0.1% (v/v) Tween-20, and 5% (w/v) nonfat dried milk. Immunodetection was performed using anti-TLR9 (Abcam), anti-aSMA (Abcam), anti-PDGFRa (Cell Signaling), antieMMP-14 (Abcam), anti-fibronectin (Abcam), anti-CD44 (Santa Cruz Biotechnology, Dallas, TX), anti-cleaved caspase-3 (Cell Signaling, Danvers, MA), and antieglyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology). The protein bands were visualized using the ECL system (Denville Scientific, South Plainfield, NJ). All Western blot densitometry data are normalized for the loading control, glyceraldehyde-3phosphate dehydrogenase.
8
Western Blotting of Cell Lysates
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Western blotting was performed as described previously (Li et al., 2011) . Briefly, the cells was lysed in RIPA buffer [50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, with the addition of Protease Inhibitor Cocktail (Roche)]. The proteins samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide resolving gels). The Antibodies used in this study were as follows: anti-GAPDH, anti-FoxP1 and anti-PDGFRa were obtained from Cell Signaling Technology, and the anti-Mouse/-Rabbit Secondary Antibodies were purchased from Santa Cruz Biotechnology.
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