Figure 2: TRIB2 protein expression correlates with AKT activation status and response to PI3K inhibition. | Nature Communications

Figure 2: TRIB2 protein expression correlates with AKT activation status and response to PI3K inhibition.

From: RETRACTED ARTICLE: TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

Figure 2

(a) TRIB2 protein levels correlate with AKT-Ser473 phosphorylation in a broad range of model in vitro models. Representative images showing 100 μg (TRIB2), 50 μg (AKT-Ser473) total protein loaded per lane separated by 10% SDS–PAGE. sc indicates scramble shRNA within the indicated cell lines. (b) Immunoblot profiles for matched isogenic TRIB2 cell lines for the AKT signalling network. High-TRIB2 expression correlated with significantly increased post-translational modification(s) of downstream AKT targets. (c) Temporal inhibition of PI3K-dependent signalling following exposure to PI3K inhibitors. Total protein lysate of 50–200 μg was loaded per lane and separated by 6–12% SDS–PAGE. (d) FOXO3a-dependent gene expression was evaluated following 12 h treatment with PI3K inhibitors. NT indicates no treatment. *P<0.05 **P<0.01, ***P<0.005 by two-way ANOVA. Data represent the±s.d. ns indicates that the comparison was not statistically significant. (e) (left) Co-immunoprecipitation of TRIB2 or total AKT1 from indicated cell lines, (500 μg total protein lysate immunoprecipitated per lane) and separated by 8–12% SDS–PAGE. (right) Total protein levels in each indicated cell line for co-IP targets (100 μg total protein loaded per lane and separated by 8–12% SDS–PAGE). (f) Representative yellow fluorescent protein (YFP) complementation microscopy analysis assay for the assessment of the interaction between AKT1/2 and TRIB2. The leucine zipper Venus1 and Venus2 (ZIPV1-V2) constructs were used as a positive control, while the combination of TRIB3 V2 and V1 are our negative control. AKT1/2 JIP1 constructs were an additional control of a known AKT protein/protein complex. BF indicates bright field. (g) (top) Percentage of YFP positive cells determined by FACS. (bottom) Mean fluorescent intensity of YFP positive cells. One-way ANOVA with Dunnett’s multiple comparison test, n=4 (*P≤0.05, **P≤0.01, ***P≤0.001). All analysis was conducted compared to zipV1trib3V2 (lane 1).

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