Fig 2: Bhd1 immunoprecipitates Vma2, a component of the V-ATPase complex, in S. pombe and S. cerevisiae and regulate its activity(A) Tables listing proteins common to one-step Bhd1-TAP (n = 2), two-step Bhd1-TAP (n = 2), one step Fnp1-TAP (n = 5) and two-step Fnp1-TAP (n = 2) purifications. The numbers in parentheses correspond to the total number peptides and percent protein coverage based on total number of amino acids, respectively, for representative purifications. See also Tables S1 and S2.(B) A representative Western blot showing that Bhd1-TAP co-precipitates with Vma2-MYC in presence (+aa) or absence (-aa) of amino acids. The images are representative of three biological replicates.(C) A representative Western blot showing that Lst7-TAP (the S. cerevisiae homolog of Bhd1) co-precipitates Vma2 in presence (+aa) or absence (-aa) of amino acids. The images are representative of two biological replicates. Untagged S. cerevisiae cells were used as a negative control.(D) Representative fluorescence microscopy images depicting vacuolar pH of WT, bhd1? and fnp1? cells grown continuously in EMM media supplemented with amino acids. FM4-64 (red) was used to stain vacuoles; DND189 (green) stains acidic organelles, such as vacuoles (pKa ~5.2). A culture of WT cells incubated with 10nM of Bafilomycin (V-ATPase-specific inhibitor) in pH 7.5 was used as positive control. Co-localization of green (acidic pH) and red (vacuole) pixels is depicted (Merge). DIC - differential interference contrast. Scale bar – 10µm.(E) Graph representing the quantification of co-localization of red (vacuoles) and green (acidic organelles) pixels. Mean and standard deviation of three independent experiments are shown. See also Figure S6.(F) Indicated strains were grown to log phase in complete media (YEA) and then spotted in 10-fold decreasing serial dilutions on YEA, YEA plus 700nM Bafilomycin and YEA pH = 9.0.