Cam xy sex
GFP alone, purified as described previously (14, 16) was provided by Kylie Wagstaff, and Ca M-GST (3) was provided by Jade Forwood.
Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain Ca M-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both Ca M and hsc70, in contrast to an SRY mutant derivative with impaired Ca M binding.
Results were expressed as the fractional recovery of Fn/c (Fn/c of respective time points divided by prebleach value), and data were fitted exponentially according to the formula ½).
The initial rate was determined using results for the Fn/c between 0 and 100 s post-bleaching.
We find that direct binding of hsc70 to SRY·Ca M dependent on Ca Plasmids encoding the GFP-SRY full length and HMG wild type and mutant derivatives of the Ca M-NLS (M64T and R76P) and the β-NLS (Y127C and R133W) for bacterial and mammalian expression, as well as GFP-TRF1, GFP-T-ag-NLS (amino acids 111–135), and GFP alone, have all been described (3, 13, 14).
-humidified atmosphere at 37 °C, in Dulbecco's modified Eagle's medium (DMEM; ICN Biomedicals, Costa Mesa, CA), supplemented with 10% heat-inactivated fetal calf serum (FCS; CSL, Ltd., Parkville, Victoria, Australia), 1 m hsc70 Stealth si RNA duplexes (Invitrogen) with the sense sequence 5′-UAAUUCUAAGUACAUUGAGACCAGC-3′) (7) or plasmid DNA expression constructs respectively, according to the manufacturer's specifications.