Figure S2.
Panel A: This panel shows immunoblot results. The first blot uses Phos-tag to detect phosphorylated Nvj1, with and without phosphatase treatment and glucose starvation (GS) for 2 days. The second blot detects Nsg2, the third detects Ypf1, and the fourth uses Tim23 as a loading control. Panel B: This panel shows multiple immunoblots. The first blot detects FLAG-tagged Ypf1, the second detects HA-tagged Nsg2, the third and fourth detect V5-tagged Hmg1 and Hmg2, respectively. The fifth blot uses Tim23 as a loading control, and the sixth uses Vac8. Samples are treated with or without GS for 2 days and with or without Endo H digestion. Panel C: This panel contains three bar graphs quantifying protein levels of Ypf1, Nsg1, and Nsg2 in wild-type (WT) and nvj1delta cells. The y-axes represent protein levels in arbitrary units (A.U.), and the x-axes compare WT and nvj1 delta. Panel D: This panel contains four bar graphs quantifying protein levels of Nvj1-GFP, Ypf1, Nsg1, and Nsg2 in various yeast strains (WT, ypf1 delta, nsg1 delta, nsg2 delta, nsg1delta nsg2 delta). The y-axes represent protein levels in A.U., and the x-axes compare different strains.
Nvj1 and Nsg2 are phosphorated in a GS-dependent manner. (A) Membrane fractions isolated from yeast cells with or without 2 days of GS treatment were treated with λ protein phosphatase. The samples were analyzed by Phos-tag or SDS-PAGE followed by immunoblotting. (B) Membrane fractions isolated from the indicated yeast cells cultured with or without 2 days of GS and those marked as Endo H+ were subjected to Endo H digestion before immunoblot analysis. (C and D) Bar graphs show quantification of protein levels from the immunoblots in Fig. 2, D and H, respectively. Source data are available for this figure: SourceData FS2.