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hsc70 is a constitutively expressed member of the hsp70 family of chaperone proteins, found in both the nuclear and cytoplasmic compartments, known to play a role in protein folding and translocation of proteins across the endoplasmic reticulum and mitochondrial membranes (11, 12).
Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics.Experiments were carried out in 5 μl containing 10 μ GFP-fusion protein, a 70-k Da Texas red dextran to assess nuclear integrity, untreated rabbit reticulocyte lysate (45 μg/μl; Promega, Madison, WI), and an ATP regenerating system (0.125 g/ml creatine phosphokinase; 30 m hsc70 (Stressgen Biotechnologies) and/or Ca M (Calbiochem, La Jolla, CA) purified protein.Image analysis was performed using NIH Image J software as described above.Finally, we demonstrate direct binding of hsc70 to the SRY·Ca M complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired Ca M binding, dependent on Ca box) family of chromatin remodeling factors play key roles in development (1), which is strongly dependent on nuclear localization efficiency (2).SRY plays a key role in mammalian sex determination within the nucleus, with 15% of all XY sex-reversed patients presenting with mutations in the ), which flank its HMG box domain (2) have been shown to impair the nuclear targeting of SRY specifically.For analysis of intranuclear mobility of SRY, results were expressed as the fractional recovery of the bleached area (Fn We previously reported Ca M-dependent nuclear import for SRY, as well as a number of other related SOX proteins, independent of the classical mode of nuclear transport through Imps (2, 3).
The heat shock cognate protein hsc70 has been previously reported to play a role in modulating the nuclear import of proteins such as SV40-T-ag and nucleoplasmin (9, 10), as well as being a Ca M-binding protein (21, 22) able to associate with nucleoporins (7).
Data were fitted to a sigmoidal curve and the Fn/c Immunoprecipitation was performed to assess association of hsc70 with SRY using GFP-TRAP® (Chromotek, Martinsried, Germany), as per the manufacturer's instructions, using 500 μg of rabbit reticulocyte lysate and 7 μg of recombinant GFP-fusion protein (as indicated) and 10 μl of equilibrated GFP-TRAP® beads.
Proteins dissociated from the beads were then subjected to SDS-PAGE (12% gel) electrophoresis and transferred to polyvinylidene difluoride membrane that was then probed with anti-hsc70 and then anti-GFP antibodies followed by goat anti-rat Ig G-HRP (Millipore, Bedford, MA) or goat anti-mouse Ig G-HRP (Millipore) before development using the Western Lighting Chemiluminescence reagent (Perkin Elmer Life Sciences).
chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (Ca M) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal.
However, the mechanism by which Ca M facilitates nuclear accumulation is unknown.
Assays to confirm the functionality of si RNA to hsc70 (100 n FRAP was performed as described previously (19).