Figure 5.
RAS-ROS-p38 Signaling Controls TTP Activity
(A) Histograms represent peak areas from extracted ion chromatograms for non-phosphorylated and phosphorylated peptides corresponding to S52 and S178 phosphosites of mouse TTP. Myc-TTP was immunoprecipitated from CT26 Myc-TTP (tet-ON) cells 1 hr after the indicated treatment. Mean ± SD of technical triplicates. Representative of two independent biological experiments.
(B) qPCR analysis of ER-KRASG12V type II pneumocytes treated in starvation medium for 24 hr. Mean ± SEM of four independent experiments.
(C) Representative flow cytometry histograms of PD-L1 surface protein expression in MCF10A ER-ΔMEKK3 cells treated in starvation medium for 1 day or 4 days. Data are representative of two independent experiments.
(D) Flow cytometry analysis of PD-L1 surface protein expression on ER-HRASG12V MCF10A cells (24 hr) and ER-HRASG12V HKE-3 cells (48 hr) after treatment in starvation medium. Data are representative of biological duplicates.
(E) qPCR analysis of CT26 cells at 2 hr or 24 hr after MK2 inhibition with PF 3644022. Mean ± SEM of two independent experiments.
(F) Sequence alignments of the conserved phosphosites (highlighted red) targeted by MK2 in mouse (Mm) and human (Hs) TTP protein.
(G) Western blotting of immunoprecipitations from CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs. Cells were treated with dox. for 24 hr before the addition of PMA or DMSO for 1 hr. Arrow indicates Myc-TTP. Data are representative of two independent experiments.
(H) qPCR analysis of CT26 TTP KO cells harboring tet-ON, WT, or phospho mutant, Myc-TTP constructs, treated with dox or vehicle for 48 hr. Data represent the mean ± SEM of two independent experiments.
∗∗p < 0.005, ∗p < 0.05. Unpaired, two-tailed Student’s t test. Abbreviations and quantities: 4-OHT, 100 nM; NAC, N-acetyl-L-cysteine, 10 mM; PMA, 200 nM; MEK inhibitor, GSK1120212, 25 nM; MK2 inhibitor PF 3644022, 1 μM; MK2 inhibitor III, 1 μM; dox., doxycycline, 1 μg/mL. See also Figure S5.