Increased osmolarity upregulates myc-CLN3 protein expression.

(A) Hyperosmolarity induced by NaCl/urea dramatically increases myc-CLN3 protein expression. BHK clone 19 myc-CLN3-expressing and BHK clone 2 UB6 empty vector-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 loaded on a 10% polyacrylamide gel and immunoblotted with a monoclonal anti-myc antibody. GM130, an integral Golgi membrane protein (130 kDa) was immunoblotted as a loading control. The immunoblot (A) is representative of 4 biological replicates. (B) Densitometric quantification of myc-CLN3 expression in hyperosmolarity induced by NaCl/urea. The mean pixel density for each band was measured in the ImageJ program. Myc-CLN3 band intensities were normalized to the corresponding GM130 (loading control) band intensities. Columns and bars represent mean ± S.E.M. (n = 4). Statistical significance was determined by one-way ANOVA with Bonferroni’s post-test. (C) Hyperosmolarity induced by sucrose also significantly increases myc-CLN3 protein expression. BHK clone 19 myc-CLN3-expressing cells were grown under isotonic (300 mOsm) or hyperosmotic conditions. Osmolarity was increased by sucrose at 100 mOsm intervals to 800 mOsm. Cell lysis and the immunoblot for myc-CLN3 were performed as described in (A). (D) Densitometric quantification of myc-CLN3 expression in hyperosmolarity induced by sucrose. Myc-CLN3 band intensities were normalized to the corresponding GM130 (loading control) band intensities, and expressed as fold increase of the isotonic control. Columns and bars represent mean ± S.E.M. (n = 3–4). The myc-CLN3 protein level observed when hyperosmolarity was induced by NaCl/urea (B) is shown for comparison. Statistical significance was determined by one-way ANOVA with Bonferroni’s post-test: **p<0.01 and ***p<0.001 as compared to the isotonic control.