Journal: Febs Letters

Article Title: Regulation of Rab5 isoforms by transcriptional and post‐transcriptional mechanisms in yeast

doi: 10.1002/1873-3468.12785

Figure Lengend Snippet: (A) Protein levels of Rab GTPases in WT and vps4∆ cells (ratio vps4∆/ WT) deduced from quantitative proteomics . *Significant protein ratio changes . n.d., not determined. (B–E) SDS/PAGE and western blot of whole cell protein lysates from logarithmically growing cells analysed with the indicated antibodies. (B) WT, vps4∆ and vps21∆ cells expressing the indicated Rab5 plasmids. Due to strong differences in expression we provide long and short western blot exposures (exp.). For quantifications see Fig. . (C) WT and vps4∆ cells expressing HA‐YPT53 and vps4‐ts or empty plasmid grown into logarithmic phase at permissive temperature (26 °C) and shifted to the restrictive temperature (37 °C) for the indicated time. *Unspecific background bands. For quantification see Fig. . (D) WT cells or the indicated mutants expressing 3xHA‐YPT53 . p(recursor) and m(ature) forms of Ape1. For quantification see Fig. . (E) vps21∆, ypt52∆, ypt53∆ cells expressing the indicated plasmids. (F) Growth of vps21∆, ypt52, ypt53∆ cells containing centromeric plasmids expressing 3xHA‐tagged VPS21, YPT52 or YPT53 from their native promoter and terminator sequences or empty vector at the indicated temperatures. (G) Life cell fluorescence microscopy of vps21∆, ypt52∆, ypt53∆ cells expressing Mup1‐GFP (after treatment with 100 μg·mL −1 l ‐methionine for 75 min) or GFP‐CPS and the indicated plasmids.

Article Snippet: TAQman gene expression assays were from Thermo Fisher ( YPT53 : Sc04158331_s1; VPS21 : Sc04165675_s1; PGK1 : Sc04104844_s1).

Techniques: Quantitative Proteomics, SDS Page, Western Blot, Expressing, Plasmid Preparation, Fluorescence, Microscopy

Journal: Febs Letters

Article Title: Regulation of Rab5 isoforms by transcriptional and post‐transcriptional mechanisms in yeast

doi: 10.1002/1873-3468.12785

Figure Lengend Snippet: (A) Quantification of YPT53 and VPS21 mRNA (normalized to stable PGK1 mRNA) from logarithmically growing WT ( vps4∆ + pRS413‐ VPS4 ) and ESCRT mutant ( vps4∆ + pRS413) cells by RT‐qPCR (∆∆C T method), n = 4. Error bars indicate standard deviation. n.s., not significant. *** P < 0.001. (B) SDS/PAGE and western blot from whole cell protein lysates of WT (BY4742) and congenic crz1∆ and gis1∆ cells in logarithmic phase and after 24 h starvation for amino acids and nitrogen sources analysed with the indicated antibodies. (C) Whole cell protein lysates from logarithmically growing WT (BY4742) cells and the indicated mutants expressing VPS4 or vps4 E233Q from plasmids analysed as in (B). (D) Whole cell protein lysates of logarithmically growing WT (BY4742) and indicated congenic deletion strains analysed as in B). *Nonspecific background bands. For quantifications see Fig. . (E) Quantification of YPT53 and VPS21 mRNA from logarithmically growing WT (BY4742) and congenic pat1∆ and xrn1∆ cells by RT‐qPCR as in A ( n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: TAQman gene expression assays were from Thermo Fisher ( YPT53 : Sc04158331_s1; VPS21 : Sc04165675_s1; PGK1 : Sc04104844_s1).

Techniques: Mutagenesis, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot, Expressing

Journal: Febs Letters

Article Title: Regulation of Rab5 isoforms by transcriptional and post‐transcriptional mechanisms in yeast

doi: 10.1002/1873-3468.12785

Figure Lengend Snippet: (A) Schematic depicting the chimeric constructs of VPS21 and YPT53 with exchanged upstream and downstream genetic regions. (B) Quantification of YPT53 mRNA normalized to stable PGK1 mRNA from logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs by RT‐qPCR ( n ≥ 3). Error bars indicate standard deviation. n.s., not significant, ** P < 0.01,*** P < 0.001 (C) SDS/PAGE and western blot of whole cell protein lysates from logarithmically growing vps21∆ or vps4∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed with the indicated antibodies. Due to strong differences in expression we provide long and short western blot exposures (exp.). For quantification see Fig. . (D) Quantification of VPS21 mRNA as in (B) ( n = 3), *** P < 0.001. (E) Whole cell protein lysates from logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed as in (C). p(recursor) and m(ature) forms of Ape1. (F) Whole cell protein lysates of logarithmically growing vps21∆ cells expressing the indicated plasmids treated with 50 μg·mL −1 cycloheximide (CHX) for the indicated times and analysed as in (C). For quantification see Fig. .

Article Snippet: TAQman gene expression assays were from Thermo Fisher ( YPT53 : Sc04158331_s1; VPS21 : Sc04165675_s1; PGK1 : Sc04104844_s1).

Techniques: Construct, Expressing, Quantitative RT-PCR, Standard Deviation, SDS Page, Western Blot

Journal: Febs Letters

Article Title: Regulation of Rab5 isoforms by transcriptional and post‐transcriptional mechanisms in yeast

doi: 10.1002/1873-3468.12785

Figure Lengend Snippet: (A) Growth of vps21∆, ypt52, ypt53∆ cells expressing the indicated constructs at the indicated temperatures. (B) Life cell fluorescence microscopy of Mup1‐GFP in vps21∆ cells expressing the indicated constructs grown into logarithmic phase and exposed to L‐methionine (100 μg·mL −1 ) for 90 min. Vac(uoles). Size bars 5 μm. (C) Life cell fluorescence microscopy of vps21∆, ypt52∆, ypt53∆ cells expressing GFP‐CPS and the indicated plasmids. Vac(uoles). Size bars 5 μm. (D) SDS/PAGE and western blot of whole cell protein lysates of logarithmically growing vps21∆ cells expressing the indicated VPS21/YPT53 chimeric constructs and analysed with the indicated antibodies.

Article Snippet: TAQman gene expression assays were from Thermo Fisher ( YPT53 : Sc04158331_s1; VPS21 : Sc04165675_s1; PGK1 : Sc04104844_s1).

Techniques: Expressing, Construct, Fluorescence, Microscopy, SDS Page, Western Blot