Table 1.
Yeast strains.
Fig 1.
Analysis of the cellular content of different 3xHA tagged proteins.
Cells of the wild-type (W303-1a), DDC1-3xHA (JCY1701), CLN2-3xHA (JCY1357), PKC1-3xHA (JCY2033), SLT2-3xHA (JCY411), RAD53-3xHA (JCY1905) and SWI5-3xHA (JCY487) strains were grown in YPD. Ddc1, Cln2, Pkc1, Slt2, Rad53 and Swi5 protein level in cell extracts was determined by western blot using specific anti-Ddc1, anti-Cln2, anti-Pkc1, anti-Slt2, anti-Rad53, anti-Swi5 or anti-HA antibodies. The ponceau staining of the membrane is shown as a loading control.
Fig 2.
Analysis of the cellular content of proteins labelled with different tags.
Cells of the wild-type (W303-1a), DDC1-3xHA (JCY1701), DDC1-6xHA (JCY1825), DDC1-GFP (JCY1661), DDC1-myc (JCY1887), CLN2-3xHA (JCY1357), CLN2-6XHA (JCY1830), CLN2-GFP (JCY1888), CLN2-myc (JCY1890), PKC1-3xHA (JCY2033), PKC1-6XHA (1891), PKC1-GFP (JCY1511), PKC1-myc (JCY1916), RAD53-3xHA (JCY1905), RAD53-6XHA (JCY1901), RAD53-GFP (JCY1903) and RAD53-myc (JCY1907) strains were grown in YPD. Protein level was determined by western blot using anti-Ddc1, anti-Cln2, anti-Pkc1 and anti-Rad53 antibodies. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control.
Fig 3.
Analysis of the stability of proteins labelled with different tags.
(A) Exponentially growing cultures of the wild-type (W303-1a), DDC1-3xHA (JCY1701), DDC1-6xHA (JCY1825), DDC1-GFP (JCY1661) and DDC1-myc (JCY1887) strains were incubated in the presence of 100 μg/mL cycloheximide. Ddc1 protein level was analysed at the indicated times after the addition of cycloheximide by western blot. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control. (B) Cln2 protein stability was analysed in wild-type (W303-1a), CLN2-3xHA (JCY1357), CLN2-6XHA (JCY1830), CLN2-GFP (JCY1888) and CLN2-myc (JCY1890) cells as described in A. (C) Pkc1 protein stability was analysed in wild-type (W303-1a), PKC1-3xHA (JCY2033), PKC1-6XHA (1891), PKC1-GFP (JCY1511) and PKC1-myc (JCY1916) cells as described in A. (D) Rad53 protein stability was analysed in wild-type (W303-1a), RAD53-3xHA (JCY1905), RAD53-6XHA (JCY1901), RAD53-GFP (JCY1903) and RAD53-myc (JCY1907) as described in A. Protein level was determined by western blot using anti-Ddc1, anti-Cln2, anti-Pkc1, anti-Rad53, anti-HA, anti-GFP or anti-myc antibodies as indicated. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control.
Fig 4.
Functional analysis of proteins labelled with different tags.
(A) 10-fold serial dilutions from exponentially growing cultures of wild-type (W303-1a), DDC1-3xHA (JCY1701), DDC1-6xHA (JCY1825), DDC1-GFP (JCY1661) and DDC1-myc (JCY1887) strains were spotted onto YPD medium and exposed to UV radiation (40 J/m2). Plates were incubated at 25°C for 4 days. (B) Cell size distribution in exponentially growing cultures of cln1 (JCY275), cln1 cln2 (JCY847), cln1 CLN2-3xHA (JCY929) and cln1 CLN2-6XHA (JCY1960) in complete SD medium. (C) 10-fold serial dilutions from exponentially growing cultures of wild-type (W303-1a), PKC1-3xHA (JCY2033), PKC1-6XHA (1891), PKC1-GFP (JCY1511) and PKC1-myc (JCY1916) were spotted onto YPD medium. Plates were incubated at 25°C, 33°C or 37°C for 3 days. (D) 10-fold serial dilutions from exponentially growing cultures of wild-type (W303-1a), RAD53-6xHA (JCY1901), RAD53-3XHA (JCY1905), RAD53-GFP (JCY1903) and RAD53-myc (JCY1907) were spotted onto YPD medium containing 200 mM hydroxyurea. Plates were incubated at 25°C for 3 days.
Fig 5.
Analysis of the effect of the spacer sequence in 3xHA-tagging modules.
(A) Schematic representation of Ddc1-3xHA, Ddc1-GFP and Ddc1-myc including the spacer sequence between the Ddc1 protein and the tag. (B) Cells of the wild-type (W303-1a), DDC1-3xHA (JCY1701) and DDC1-3xHAΔIF (JCY2063) strains were grown in YPD. Ddc1 protein level in cell extracts was determined by western blot using a specific anti-Ddc1 antibody. The ponceau staining of the membrane is shown as a loading control. (C) Exponentially growing cultures of the wild-type (W303-1a), DDC1-3xHA (JCY1701) and DDC1-3xHAΔIF (JCY2063) strains were incubated in the presence of 100 μg/mL cycloheximide. Ddc1 protein level was analysed at the indicated time after the addition of cycloheximide by western blot using an anti-Ddc1 antibody. Cdc28 recognized by the anti-PSTAIRE antibody is shown as a loading control. (D) 10-fold serial dilutions from exponentially growing cultures of wild-type (W303-1a), DDC1-3xHA (JCY1701) and DDC1-3xHAΔIF (JCY2063) strains were spotted onto YPD medium and exposed to UV radiation (40 J/m2). Plates were incubated at 25° for 4 days. (E) Whi7 was tagged with 3xHA using either the transforming modules described in Longtine et al. (JCY1544, lane 1) or an alternative cassette (JCY1728, lane 2). Whi7 protein level in cell extracts was determined by western blot using an anti-HA antibody. Note that Whi7 migrates in SDS-PAGE as multiple bands, which correspond to distinct phosphorylated states. The ponceau staining of the membrane is shown as a loading control.