Fig. 2. Cln2 is located in both the nucleus and the cytoplasm. Strain N-5 containing an empty plasmid (
) or the plasmid pSL46(GAL::CLN2-3xHA) (HA) was grown at 30°C in selective media containing 2% raffinose to 5x106 cells/ml. The culture was divided in two, and galactose was added to one of them (1% final). All four cultures (+/– CLN2-3xHA, and +/– galactose induction) were grown for an additional hour then collected and fractionated. (a) Cytoplasmic and nuclear fractions from induced cln2 or GAL-CLN2-3xHA cells were analyzed using SDS/PAGE and blotted onto a membrane. 20 µg of protein from the cytoplasmic fraction, and 20 µg of protein from the nuclear fraction, were loaded. The western blot was first probed with anti-HA 12CA5 monoclonal antibody to detect Cln2, and subsequently reprobed with an anti-ADH polyclonal antibody as a cytoplasmic control, and a monoclonal antibody that recognizes the nuclear protein Nop1 (a kind gift from J. Aris, University of Florida, FL). (b) Cytoplasmic and nuclear fractions from uninduced (–) or induced (+) cells bearing GAL-CLN2-3xHA were analyzed using SDS-PAGE and blotted onto a membrane. 7 µg of protein was loaded. The western blot was probed with an anti-Sic1 polyclonal antibody (kindly provided by J. Donovan, SUNY at Stony Brook, NY). The top panel shows a high molecular weight nonspecific band as an internal loading control. (c) Plasmid pSL122(GAL::CLN24T3S-3xHA) was introduced to strain N-5, and treated as in (a). Equal amounts of protein were loaded in each lane. The blot was probed with anti-HA (12CA5) antibody to detect Cln2-3xHA, and subsequently reprobed with an anti-ADH polyclonal antibody, and anti-NOP1 monoclonal antibody.
