Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

Supplementary Figure

Files in this Data Supplement:

  • Supplementary Figure 1 - FSH stimulates protein neosynthesis as a function of cell density in HEK293-FSHR. HEK293-FSHR cells were seeded at increasing density, as indicated, prior to stimulation with 100 ng/ml FSH along with 35S-labeled Methionine and Cysteine. Ninety minutes later, cells were harvested and radiolabeled proteins were quantitated as trichloroacetic (TCA) precipitable counts per minute. The total radioactivity incorporated in unstimulated versus FSH-stimulated HEK293-RFSH cells was compared. Results are expressed as means +/- SEM of triplicates.(PDF 45 KB)
  • Supplementary Figure 2 - Analysis of 4E-BP1 phosphorylation profile. HEK293-FSHR lysates from time-course of FSH stimulation were analyzed by Western blot. A. C. E. The phosphorylation of 4E-BP1 was detected using specific antibodies raised against phospho-Thr70, phospho-Thr37/Thr46 and phospho-Ser65 respectively, as indicated. Membranes were reprobed with an anti-GAPDH or anti-vinculin antibody as indicated, to monitor gel loading. B. D. F. Densitometry analysis of Western blots probed with antibodies raised against 4E-BP1 phosphorylated on Thr70 (n=6), Thr37/Thr46 (n=5) and Ser65 (n=7) respectively. Data are expressed as % of the maximal response in at least 5 experiments, and the points represents the means +/- SEM of these values. One hundred percent was not reached in case the timepoint of the maximum response varies. Statistical significance was calculated using one-way ANOVA with a Bonferroni post-test in control versus FSHstimulated cells. (PDF 228 KB)
  • Supplementary Figure 3 - Cap-colum experiments to detect 4E-BP1 (A) or eIF4G (B) by Western blot, upon 60 or 30 min of cell stimulation with 20 % FBS (n=3). Membranes were reprobed with anti-eIF4E antibody to monitor the input. WCL = whole cell lysate. (PDF 73 KB)
  • Supplementary Figure 4 - FSH stimulates protein neosynthesis in a rapamycin-sensitive manner. HEK293-FSHR cells were preincubated with 10 nM rapamycin for one hour prior to 35S-labelling and stimulation with FSH for 90 min. Radiolabeled proteins were quantitated as TCA-precipitable counts per minute. Raw count data in TCA precipitates were normalized with the quantity of total proteins, and are expressed as means +/- SEM of percentage of FSH-induced radiolabeled aminoacid incorporation, taken as 100 %, in 3 independent experiments. (PDF 37 KB)
  • Supplementary Figure 5 - HEK293-FSHR cells were transfected with plasmids expressing cap-dependent renilla luciferase and VEGF IRESB-dependent firefly luciferase. Forty-eight hours after transfection, cells were serum-starved for 2 hours, prior to stimulation for 24 hours with increasing concentrations of FSH. Results are expressed as relative light units of VEGFB IRES- and cap-driven luciferase activities. The graph shown represents the means of six values obtained from 2 independent experiments ± SEM. (PDF 40 KB)

This Article

  1. J Mol Endocrinol June 1, 2014 vol. 52 no. 3 373-382
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  2. Supplementary Figure