Tunicamycin and TCA were from Sigma-Aldrich (St. Louis, MO), brefeldin A was from Epicentre (Madison, WI), and ionomycin and rapamycin were from Calbiochem (San Diego, CA). PMA was from Sigma-Aldrich, and EGF receptor inhibitor AG1478 was from Cell Signaling (Danvers, MA). Polyclonal antibodies against GFP and cyclophilin B were from Invitrogen (Carlsbad, CA) and Abcam (Cambridge, MA), respectively. The polyclonal fibronectin antibody was from Millipore (Billerica, MA). Alexa Fluor 568–coupled secondary antibody was purchased from Life Technologies (Invitrogen).
Ag1478
Manufactured by Cell Signaling Technology
Sourced in United States
AG1478 is a tyrosine kinase inhibitor that selectively inhibits the epidermal growth factor receptor (EGFR) kinase. It functions by blocking EGFR autophosphorylation and inhibiting EGFR-mediated signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin antibody, by Merck Group (1 mentions)
Brefeldin a, by Illumina (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Triphenyl tetrazolium chloride (ttc), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by Wuhan Boster Biological Technology (1 mentions)
Human recombinant tnfα, by R&D Systems (1 mentions)
Rabbit monoclonal anti egfr antibody d38b1, by Cell Signaling Technology (1 mentions)
Rabbit anti β actin polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Normal armenian hamster igg, by Santa Cruz Biotechnology (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Rapamycin, by Fujifilm (1 mentions)
Dynasore, by Adipogen (1 mentions)
Af6000, by Leica (1 mentions)
Torin 1, by Cayman Chemical (1 mentions)
Nocodazole, by Adipogen (1 mentions)
Mouse anti ki67 antibody, by BD (1 mentions)
Click it edu detection, by Thermo Fisher Scientific (1 mentions)
Sar 405, by Cayman Chemical (1 mentions)
6 protocols using ag1478
1
Immunofluorescence Assay with Inhibitors
2
Dex-Induced Astrocyte Activation and EGFR Signaling
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Dex was manufactured and supplied by Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China). Rat glial fibrillary acidic protein (GFAP; cat. no. P90068Hu01) and recombinant rat ADRA2A monoclonal antibodies (cat. no. CSB-YP001388MO) were purchased from Shanghai Yanhui Biotechnology Co., Ltd. (Shanghai, China) and Wuhan Huamei Biotechnology Co., Ltd. (Beijing, China) and used for immunofluorescent staining and protein blotting during the following experiments. Goat anti-rabbit (cat. no. BA1055) or goat anti-rat (cat. no. BA1058) horseradish peroxidase-conjugated secondary antibodies and a bicinchoninic acid (BCA) protein assay kit were purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, China). Anti-β-actin rabbit monoclonal antibody (cat. no. 4970), rabbit ERK1/2 (cat. no. 4348) and phosphorylated (p)-ERK1/2 (Thr202/Tyr204; cat. no. 9101) antibodies, and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Triphenyl tetrazolium chloride (TTC) was acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
3
Immunoblotting Analysis of Receptor Tyrosine Kinases
4
Quantifying Neural Stem Cell Proliferation
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To monitor NSC proliferation, we analyzed NSCs by immunostaining of mouse anti–Ki-67 antibody (BD Pharmingen) and Click-IT EdU detection (Invitrogen) after 4 h exposure to EdU, and co-stained with rabbit anti-Sox2 antibody (EMD Millipore) or DAPI (Sigma) to count all cells. Cell images were obtained on AF6000 (Leica) and BZ-X (Keyence). Cell counting was performed using the ImageJ software for images captured on the AF6000, and Hybrid cell count (Keyence), an algorithm for cell counting, for images captured on the BZ-X. Six to eight images were collected under each condition. Inhibitors used for cell culture were obtained from the indicated suppliers: bafilomycin A1 (Sigma), SAR405 (Cayman Chemical), Dynasore (Adipogen), thapsigargin (Adipogen), nocodazole (Adipogen), DAPT (Calbiochem), AG1478 (Cell Signaling Technology), Torin-1 (Cayman Chemical), and rapamycin (Wako). To measure protein stability, cells were incubated with 10 µg/ml cycloheximide (Sigma) to block protein synthesis.
5
GPCR Signaling Modulation Assay
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The following drugs were used in this study: pertussis toxin (PTX; List Biological Laboratories Inc.); AG1478 (Cell Signaling Technology); ZCL278, EHT1864 (Cayman Chemicals); SP600125, Y27632 (Wako); NSC23766, SB203580, and Akti1/2 (Abcam). MCHR1:EGFP and SSTR3:EGFP clone cells were serum‐starved for 24 h prior to the pretreatment. Cells were pretreated with 120 ng/ml PTX for 24 h in serum‐starved medium. For other reagents, the final concentrations of the reagents and the pretreatment periods were: 10 µM AG1478 (30 min); 10 µM Y27632 (30 min); 50 µM ZCL278 (30 min); 10 µM EHT1864 (30 min); 3 µM NSC23766 (30 min); 30 µM SB203580 (30 min), 3 µM SP600125 (30 min), and 3 µM Akti1/2 (30 min).
6
Virus Quantification and Infectious Center Assays
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Confluent fibroblasts were infected with 0.5 MOI of WT virus and were treated with DMSO, 5μM AG1478 (Caymen Chemical), or 20μM LY294002 (Cell Signaling) at 18 hpi. Virus present in cells and media were quantified by the TCID50 as described previously [10 (link)]. An infectious centers assay was used to quantitate infectious centers produced by CD34+ HPCs, as described previously [75 (link)]. CD34+ HPCs were treated with 10 μM AG1478, 10μM Gefitinib (Cell Signaling), 20 μM LY294002 or DMSO for vehicle control.
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