Osmotic Stress Changes the Expression and Subcellular Localization of the Batten Disease Protein CLN3
Figure 3
Glycosylation of CLN3 does not appear affected under increasing osmolarity.
BHK clone 19 myc-CLN3-expressing cells were grown under isotonic (300 mOsm) or hyperosmotic conditions. Osmolarity was increased at 100 mOsm intervals to 500, 600 or 800 mOsm by the addition of NaCl plus urea (1.5∶1 molar ratio). After being exposed to 500, 600 or 800 mOsm for 24 hours, cell lysates were prepared using 1% DDM detergent under non-denaturing conditions. Twenty-five-µg protein from each sample was treated with the N-glycosylase, PNGase F. PNGase F-treated and untreated protein samples were loaded on a 10% polyacrylamide gel and immunoblotted with a monoclonal anti-myc antibody. GM130, an integral Golgi membrane protein of 130 kDa was immunoblotted as a loading control. Treatment with PNGase F resulted in the same lower molecular weight bands (40–45 kDa) at 300, 500, 600 and 800 mOsm.