Fig. S3.

Cellular concentrations of V-1 and CP, V-1 immunostaining, FCS, quantitation of V-1 overexpression, and CAR1 levels. (A, Upper) Western blot of a Dictyostelium whole cell extract (WCE; corresponding to 5 × 10⁵ amoebas) and the indicated amounts of purified, FLAG-tagged D.d. V-1 (in ng) probed with the antibody to D.d. V-1. Molecular mass markers are indicated. (A, Lower) Intensities (in A.U.) of the purified V-1 bands, as determined by densitometry using Image J2, and plotted vs. the amount of V-1 loaded (in ng). The resulting standard curve was obtained by linear regression not anchored at 0. The content of V-1 in the WCE band was estimated from this standard curve as indicated. (B, Upper) Western blot of a Dictyostelium WCE (corresponding to 1 × 10⁵ amoebas) and the indicated amounts of purified D.d. CP (in ng) that was probed with the antibody to D.d. CP. Molecular mass markers are indicated. (B, Lower) The intensities (in A.U.) of the purified CP bands, as determined by densitometry using Image J2, and plotted vs. the amount of CP loaded (in ng). The resulting standard curve was obtained by linear regression not anchored at 0. The content of CP in the WCE band was estimated from this standard curve as indicated. (C) A field of WT Dictyostelium amoeba fixed and stained for V-1. (Scale bar, 10 µm.) (D) A field of V-1–null cells fixed and stained for V-1 and imaged using the exact same parameters as in C. (Scale bar, 10 µm.) (E) A representative FCS plot for mRFP in the cytoplasm. (F) A representative FCS plot for mRFP-V-1 in the cytoplasm. (G) Western blot of WCEs made from cells that were expressing mRFP V-1 at L, M, and H levels (fractionated by a fluorescence activated cell sorter as described in Methods). The blot was probed with the antibody to D.d. V-1. For L, M, and H expressers, the ratio of mRFP V-1 to endogenous V-1 was 0.45 ± 0.07, 1.55 ± 0.07, and 3.00 ± 0.42, respectively. (H) As in G, except that the cells were expressing mRFP FBM-V-1. For L, M, and H expressers, the ratio of mRFP FBM-V-1 to endogenous V-1 was 0.65 ± 0.07, 1.95 ± 0.09, and 3.50 ± 0.28, respectively. Note that V-1 overexpression did not result in a lowering of the level of endogenous V-1. (I) Western blot of WCEs prepared from vegetative WT cells (lane 1), vegetative V-1–null cells (lane 2), ripple stage WT cells (lane 3), and ripple stage V-1–null cells (lane 4) and probed with a monoclonal antibody to the cAMP receptor CAR1.