Figure 6. The MASTL‐PP2A/B55 pathway controls the feedback loop at the level of adaptor proteins.

1. MDA‐MB‐231 cells were co‐transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phosphoresidues. Plots show mean + SEM from three independent experiments. *P < 0.05, Student's t‐test.
2. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re‐stimulated with glucose for 15 min.
3. siRNA‐mediated genetic depletion of B55 alpha and delta subunits (B55αδ) in control (Dox −) and MASTL‐null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re‐addition. n = 3 independent experiments. The plot shows mean + SEM from three independent experiments. *P < 0.05, one‐way ANOVA.
4. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1 and phospho‐S6K1 T389 [normalized to total S6K1 level; n = 6 Mastl(+/+) and 7 Mastl(Δ/Δ)]. β‐Actin or vinculin was used as a loading control. Data are mean ± SEM; *P < 0.05, unpaired Student's t‐test.