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Fig. 3 | Molecular Medicine

Fig. 3

From: GTK Tyrosine Kinase-induced Alteration of IRS-protein Signalling in Insulin Producing Cells

Fig. 3

Insulin-induced IRS-1 and IRS-2 phophorylation in RINm5F cells. Serum-starved control (neo-1 and neo-2) RINm5F cells or RINm5F cells overexpressing GTK (GTKwt, GTKY394F and GTKY504F) were stimulated with insulin as described above. The cells were lysed, immunoprecipitated for IRS-1 or IRS-2 and subjected to Western blot analysis for phosphotyrosine (P-Tyr), IRS-1, IRS-2 and SHB. (A). Tyrosine phosphorylated IRS-1 was detected as a 185 kDa band (top). Densitometric scanning of tyrosine-phosphorylated and total amount of IRS-1 and the mean IRS-1 phosphorylation normalised against the total amount of IRS-1 protein was calculated (middle). The stimulation of IRS-1 phosphorylation in response to insulin was calculated as the percent of maximum stimulation, the largest fold increase of IRS-1 phosphorylation in each experiment was set to 100% (bottom) (B). Tyrosine phosphorylated IRS-2 was detected as a 200 kDa band (top). Densitometric scanning of tyrosine-phosphorylated and total amount of IRS-2 and the mean phosphorylation normalised against the total amount of IRS-2 was calculated (middle). The stimulation of IRS-2 phosphorylation in response to insulin was calculated as the percent of maximum stimulation as described above (bottom). Bars are means ± SEM from 4–5 independent experiments. *, p < 0.05 and **, p < 0.01 when compared with corresponding value of RINm5F-control (neo).

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