Kinase assay - GRPR activation
Description
A kinase assay was carried out using the STK pamgene kinase assay to understand what was the signaling induced by the activation of GRPR. 1057 mouse melanoma cells were stimulated for 15 minutes with 10 nM GRP and/or 1µM RC-3095 in F12 0.5% FBS. Proteins were extracted with M-Per buffer supplemented with Halt protease Inhibitor cocktail (Thermo Scientific #78430) and Halt phosphatase inhibitor cocktail (Thermo Fisher #78442) and immediatly snap-frozen. 1 µg of proteins were analysed using STK chips and ran according to the manufacturer recommendation. Four biological replicates were generated Groups: - MOCK = 0.1% DMSO - GRP = 10nM GRP in 0.1% DMSO - RC = 1µM RC-3095 in 0.1% DMSO - MOCK = 10nM GRP + 1µM RC-3095 in 0.1% DMSO
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Steps to reproduce
Cells 500,000 cells were seeded in a 6 well plates. The next day, the cells were starved overnight (18h) in 0.5% FBS media. Cells were stimulated in 0.5% FBS with the different treatments for 15 minutes at 37°C: - Mock = 0.1% DMSO - GRP = 10nM GRP[18-27] (Bachem #4011670) in 0.1% DMSO - RC = 1µM RC-3095 (Sigma #R9653) in 0.1% DMSO - GRP + RC = 10nM GRP[18-27] and 1µM RC-3095 in 0.1% DMSO After stimulation, cells were washed with cold PBS and lysed in M-Per buffer (Thermo Scientific #78501) supplemented with Halt protease Inhibitor cocktail (Thermo Scientific #78430) and Halt phosphatase inhibitor cocktail (Thermo Fisher #78442). Insoluble fraction was removed by centrifugation (10,000g, 15 minutes at 4°C) and supernatants were snap frozen with a part saved to quantify proteins. Protein quantification was performed by BCA. 1µg of protein lysate was ran on a PamGene STK Chip according to manufacturer recommendations using the Pamstation PS12. The analyses were performed using the bionavigator software provided by PamGene. First batch were corrected using ComBat and then data were normalised using VSN. Normalised data were used to compute the kinase activity using the 2018 version of the UKA tool. (Scan rank from 4 to 12, 500 permutations, 90% homology, equivalent weight for each database, minimal prediction score of 300)
Institutions
- Institut Curie Centre de Recherche