Total proteins were harvested from tissue and cell lines lysates using M-PER protein extraction reagent (Thermo Fisher, USA) respectively supplemented with proteinase and phosphatase inhibitors (Sigma, USA) according to standard protocol. Protein concentrations were determined using the BCA method followed by standard western immunoblotting of proteins using different primary antibodies: Anti-METTL3 (#195352, abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), Anti-FTO (#ab124892, abcam, USA), Anti-P21 (#ab109199, abcam, USA), Anti-Phospho-AKT (#9271, Cell Signaling, USA), Anti-AKT (#9272, Cell Signaling, USA), Anti-Phospho ATM (#4526, Cell Signaling, USA), Anti-ATM (#2873, Cell Signaling, USA), Anti-Phospho-IR/IGF1R (#3021, Cell Signaling, USA), Anti-IGF1Rβ (#9750, Cell Signaling, USA), Anti-IRβ (#3025, Cell Signaling, USA), Anti-PDX1 (#5579, Cell Signaling, USA), Anti-Pan-Calcineurin A (#2614, Cell Signaling, USA), Anti-SHP-2 (#3397, Cell Signaling, USA), Anti-β-Actin (#4970, Cell Signaling, USA), Anti-αTubulin (#7291, abcam, USA). (Please see Reporting Summary for further details on antibodies used). The blots were developed using chemiluminescent substrate ECL (ThermoFisher, USA) and quantified using Image studio Lite Ver. 5.2 software (LICOR, USA).
Anti pdx1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PDX1 is a primary antibody that recognizes the pancreatic and duodenal homeobox 1 (PDX1) protein. PDX1 is a transcription factor that plays a critical role in the development and function of pancreatic beta cells. This antibody can be used to detect and analyze the expression of PDX1 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti insulin, by Cell Signaling Technology (4 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Proteinase and phosphatase inhibitors, by Merck Group (2 mentions)
M per protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Anti mettl14, by Merck Group (2 mentions)
Anti α tubulin, by Abcam (2 mentions)
Anti mettl3, by Abcam (2 mentions)
Anti fto, by Abcam (2 mentions)
Anti p21, by Abcam (2 mentions)
Anti phospho atm, by Cell Signaling Technology (2 mentions)
Anti phospho ir igf1r, by Cell Signaling Technology (2 mentions)
Anti igf1rβ, by Cell Signaling Technology (2 mentions)
Anti irβ, by Cell Signaling Technology (2 mentions)
Anti pan calcineurin a, by Cell Signaling Technology (2 mentions)
Anti shp2, by Cell Signaling Technology (2 mentions)
Image studio lite ver 5, by Thermo Fisher Scientific (2 mentions)
Anti mafa, by Cell Signaling Technology (2 mentions)
10 protocols using anti pdx1
1
Quantitative Protein Analysis in Cell Lines
2
Protein Expression Analysis in BRIN-BD11 Cells
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BRIN-BD11 cells were lysed using TENNS lysis buffer [120 mM of NaCl, 1% Triton X-100, 20 mM of Tris–HCl pH 7.5, 10% glycerol, 2 mM of EDTA and protease inhibitor cocktail (10 μg/mL each of aprotinin and leupeptin)]. Approximately 30 μg of protein was resolved by 10% SDS-PAGE and a Western blot analysis was carried out using specified antibodies. The catalogue and primary dilution of the antibodies used in the study are Anti-PIMT (Abcam, Cambridge, UK, catalogue no. Ab70559) 1:2000; anti-PDX1 (Cell Signaling Technologies, Danvers, MA, USA, catalogue no. 5679) 1:1000; anti-MafA (sc-390491) 1:200); anti-HDAC5 (sc-133225) 1:1000; anti-GCK (sc-17819) 1:200; anti-insulin (Cell Signaling Technologies, 8138) 1:1000; anti-Synaptotagmin13 (Novus Biologicals, NB2-20546) 1:1000; anti-SNAP25 (sc-390644 Santa Cruz Biotechnology, Finnell Street Dallas, TX, USA) 1:200; anti-Kir6.2 (sc-20809) 1:200; anti-NeuroD1 (Cell Signaling Technologies, 2833) 1:1000; and anti-GAPDH (Cell Signaling Technologies, 2118) 1:5000. The protein levels were normalized using GAPDH and Western blots were quantified by using ImageJ [32 (link)].
3
Immunofluorescence Staining of Pancreatic Markers
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Immunofluorescence staining was performed as previously reported10 (link). The following primary antibodies were used: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-C-peptide (dilution 1:100, #4593S; Cell Signaling Technology), anti-PDX-1 (dilution 1:200, #5679; Cell Signaling Technology), anti-NKX6.1 (dilution 1:400, #54551; Cell Signaling Technology), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech, Chicago, IL, USA), anti-GAD65 (dilution 1:50, ab239372, Abcam plc.) and anti-PD-L1 (dilution 1:25, 17952–1-AP, Proteintech). Anti-rabbit Alexa 488 (A-11008; Thermo Fisher Scientific Inc.) for anti-C-peptide, PDX-1, ICA, ZnT8, GAD65 and PD-L1 antibodies, and anti-mouse Alexa 594 (A-11005; Thermo Fisher Scientific Inc.) for anti-insulin antibody were used as the secondary antibodies. 4′, 6-diamidino-2-phenylindole (DAPI) (P-36931; Thermo Fisher Scientific Inc.) was applied for nucleus staining.
4
Comprehensive Immunohistochemical Profiling of Pancreatic Cells
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Immunohistochemical staining was performed as previously reported56 (link) with the following antibodies: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-PDX-1 (dilution 1:50, #5679; Cell Signaling Technology), anti-PD-L1 (dilution 1:100, 17952–1-AP, Proteintech), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech), anti-GAD65 (dilution 1:2000, ab239372; Abcam plc.), anti-CD4 (dilution 1:1000, ab183685; Abcam plc.) and anti-CD8 (dilution 1:2000, ab217344; Abcam plc.).
5
Western Blot Analysis of Pancreatic Cell Markers
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Collected SHED and SHED-β cells were lysed in ice-cold RIPA buffer containing protease inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA). The same amount of proteins was resolved on the SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane using an electroblot. After blocking with 5% milk TBS-T, the membrane was incubated with anti-PDX1, anti-β-actin (Cell Signaling Technology Inc., Beverly, MA, USA), anti-NEUROG3, anti-ARX, anti-GLUT2 (Sigma-Aldrich, St. Louis, MO, USA), anti-NKX6.1 (R&D Systems, Minneapolis, MN, USA), anti-PAX4 (GeneTex Inc., Irvine, CA, USA), and anti-ZIP8 (Thermo Scientific Inc., Rockford, IL, USA) antibodies. A horseradish peroxidase-conjugated secondary antibody was added, and chemiluminescent reagents were used to detect immunoreactive proteins on X-ray films. A density measurement was performed on Multi Gauge V3.0 (Fujifilm, Tokyo, Japan), and the quantities were calculated by subtraction between ZIP8 and β-actin bands.
6
Immunostaining of E9.5 Mouse Embryos
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E9.5 mouse embryos were processed according to the published iDisco protocol [34 (link)]. Primary antibodies were incubated for 5 days, secondary antibodies for 4 days at 37 °C. The following primary antibodies were used for staining: anti-GFP (goat 1:500, Biotrend, Köln, Germany; 600-101-215), anti-Foxa2 (mouse 1:500; Millipore, Darmstadt, Germany, 17-10258), anti-Pdx1 (rabbit 1:500; Cell Signaling, Danvers, MS, U.S.A.; D59H3), and secondary antibodies: anti-goat 488 (1:800; Thermo Scientific, Darmstadt, Germany, A11055), anti-mouse CY5 (1:800; Dianova, Hamburg, Germany; 715-175-151) and anti-rabbit 555 (1:800; Thermo Scientific, Darmstadt, Germany, A31572). Images were taken using tile scan mode on a Zeiss LSM 880 using ZenBlack software 2.0.
7
Pancreatic Cell Protein Expression
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The SDF-1α, Arx, Pax4, Insulin and Glucagon expression were assessed by western blotting analysis and samples were normalized to GAPDH. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4°C overnight with anti-SDF-1α (1:1000, Santa cruz), anti-Arx (1:1000, Santa cruz), anti-Pax4 (1:1000, Santa Cruz), anti-Insulin (1:1000, Santa cruz), anti-Glucagon (1:1000, Santa cruz), anti-Aldh1a3 (1:1000, Novus), anti-Neurog3 (1:1000, Beta Cell Biology Consortium), anti-MafA (1:1000, Cell Signaling Technology), anti-Pdx1 (1:1000, Cell Signaling Technology), anti-NeuroD1 (1:1000, Cell Signaling Technology) and anti-XBP1 (1:1000, Santa cruz), anti-CD63 (1:2000, Abcam), anti-TSG101 (1:1000, Santa Cruz), anti-Ago2 (1: 1000, Santa Cruz) and anti-GAPDH (1:3000, Santa Cruz) antibodies respectively.
8
Quantitative Protein Analysis in Cell Lines
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Total proteins were harvested from tissue and cell lines lysates using M-PER protein extraction reagent (Thermo Fisher, USA) respectively supplemented with proteinase and phosphatase inhibitors (Sigma, USA) according to standard protocol. Protein concentrations were determined using the BCA method followed by standard western immunoblotting of proteins using different primary antibodies: Anti-METTL3 (#195352, abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), Anti-FTO (#ab124892, abcam, USA), Anti-P21 (#ab109199, abcam, USA), Anti-Phospho-AKT (#9271, Cell Signaling, USA), Anti-AKT (#9272, Cell Signaling, USA), Anti-Phospho ATM (#4526, Cell Signaling, USA), Anti-ATM (#2873, Cell Signaling, USA), Anti-Phospho-IR/IGF1R (#3021, Cell Signaling, USA), Anti-IGF1Rβ (#9750, Cell Signaling, USA), Anti-IRβ (#3025, Cell Signaling, USA), Anti-PDX1 (#5579, Cell Signaling, USA), Anti-Pan-Calcineurin A (#2614, Cell Signaling, USA), Anti-SHP-2 (#3397, Cell Signaling, USA), Anti-β-Actin (#4970, Cell Signaling, USA), Anti-αTubulin (#7291, abcam, USA). (Please see Reporting Summary for further details on antibodies used). The blots were developed using chemiluminescent substrate ECL (ThermoFisher, USA) and quantified using Image studio Lite Ver. 5.2 software (LICOR, USA).
9
Immunofluorescence Analysis of Pancreatic Islets
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Isolated islets were obtained from 8–12-week-old male Balb/c mice (purchased from Experimental Animal Center, Zhejiang University, China, GradeI, Certificate No. 2008-0016) according to the protocol.47 (link) After digesting from pancreases, islets were cultured on cover slips. Both ES cell lines on their terminal differentiation day and isolated islets were fixed with cold methanol for 10 min at −20 °C. Fixed cells were blocked with 10% FBS for 1 h at room temperature. After that, cells were incubated at 4 °C overnight with primary antibodies: anti-insulin (1 : 100), anti-PPARα (1 : 100), anti-PPARβδδ (1 : 100), anti-PPARγ (1 : 100) or anti-Pdx-1 (1 : 100, Cell Signaling Technology). Cultures were treated with appropriate secondary antibodies (1 : 400) for 2 h and DAPI (2 μg/ml, Sigma Aldrich) for 1 min at room temperature. Finally, differentiated cells were observed under Leica DMI3000B microscope (Leica, Mannheim, Germany), and isolated islets were observed under Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan). The overlay images were merged by software Image-Pro Plus.
10
Comprehensive Pancreatic Marker and Signaling Pathway Antibody Panel
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Pancreatic marker antibody set (anti–α-amylase, anti-PDX1, and anti-PLA2GB), anti-ERK, anti-p38, anti-AKT, anti–phospho-AKT, anti-NFκB, anti–phospho-NFκB-p65, anti–E-Cadherin, anti–Cytokeratin-8, anti-vimentin, anti-PCNA, anti-PTEN, and anti–β-actin antibodies were procured from Cell Signaling technology (Danvers, MA). Anti-VEGF antibody was purchased from Santa Cruz laboratories (Dallas, TX). Anti–K-ras-GTP and anti–K-ras antibodies were purchased from NewEast Biosciences Labs (King of Prussia, PA). Anti-ISX antibody was purchased from Novus biologicals (Littleton, CO). Anti–acetylated-α-tubulin antibody was purchased from Sigma (St. Louis, MO). Reporter plasmids pGL3-VEGF and pGL3-MMP2 were obtained from a plasmid repository (Addgene, Cambridge, MA).
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