DEV035238 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Concentration-response curves for cGi500 for cGMP and cAMP, measured in vitro using lysates from HEK293A cells expressing the sensor. Twenty-four hours after calcium phosphate transfection, the cells were suspended in 5 mM Tris, 2 mM EDTA (pH 7.4), disrupted with an Ultra-Turrax homogenizer (IKA-Werke, Staufen, Germany) and centrifuged for 20 minutes at 80,000 rpm (see Nikolaev et al., 2004). Fluorescence emission spectra of the supernatant were measured as described in Materials and methods. The EC₅₀ value for cGMP is 345±16 nM (mean±s.e.m., n=3).

• Supplemental Figure S2 -

  Fig. S2. The cyclic nucleotide sensors do not alter cAMP or cGMP concentrations in the oocyte. (A) The change in the YFP/CFP emission ratio after injecting 1 mM cAMP into preantral follicle-enclosed oocytes was the same for oocytes containing 5 µM or 2 µM Epac2-camps300. Preantral follicles (140-180 µm diameter) were used for these experiments because they provided a technically easier system. (B,C) The cGMP concentration measured in antral follicle-enclosed oocytes did not depend on the amount of cGi500 protein that was expressed following RNA injection. Measured cGMP values were plotted as a function of the laser power required to obtain a CFP intensity of ∼110-140 units. Lower laser power indicates that the oocyte image was brighter, corresponding to more cGi500 protein expression (although differential absorption of light by the follicle, which depends on the depth of the oocyte in the follicle, is another contributing factor). Data points correspond to the data sets shown in Fig. 5C, for no LH (B), or for 1-1.3 hours LH treatment (C).

• Supplemental Figure S3 -

  Fig. S3. Epac2-camps300 at 5 µM, or an approximately similar concentration of cGi500, did not change the kinetics of NEBD in response to LH. (A) Epac2-camps300. (B) cGi500.

• Supplemental Figure S4 -

  Fig. S4. U0126 inhibits LH-stimulated phosphorylation of MAP kinase in mouse antral follicles. Follicles were incubated under three different conditions: no treatment; 1 hour with LH; or 1 hour with 10 µM U0126 followed by 1 hour with 10 µM U0126 plus LH. Follicle proteins were separated by SDS-PAGE and immunoblotted for phospho-MAP kinase. pMAPK band intensities were quantitated using a LICOR Odyssey system. Values obtained with LH, or U0126 plus LH, treatment were divided by values obtained with no treatment, and the ratios are presented as the mean±s.e.m. These results were reported by Norris et al. (Norris et al., 2008), but without the quantitation shown here. Methods are described by Norris et al. (Norris et al., 2008).