TABLE I. Intracellular distribution of ³⁵S-proteoglycans in corneal fibroblasts.

Corneal fibroblasts were plated at 15,000 cells/cm² on fibronectin coated plates in serum free medium [Hsia et al., 2003; Richardson et al., 2001] and allowed to incubate for 16h at 37°C. ³⁵SO₄ (100 μCi/mL) was added and the cells allowed to incubate for an additional 24 h at 37°C. The ³⁵SO₄-labeled cells were suspended with trypsin and ruptured by hypotonic shock and with NP-40. Cell extracts were separated into cytoplasmic and nuclear fractions as described in Experimental Procedures. Samples were subjected to cationic nylon filtration to separate ³⁵S-proteoglycan from unincorporated ³⁵SO₄. Duplicate filters were subjected to nitrous acid treatment [Rapraeger and Yeaman, 1989]. ³⁵S-heparan sulfate was calculated by subtracting the nitrous acid resistant ³⁵S-proteogylcan (non-HSPG) from the total PG. Intracellular PG represented ~40% of the total cell-associated PG in these cells, and isolation and quantitation of cell-associated PG generated ~1.5 μg PG/10⁶ cells (based on total GAG using the DMB assay [Farndale et al., 1986]). Hence, estimates of cytoplasmic and nuclear PG levels (μg/10⁶ cells) were calculated as: 1.5*0.4*(relative amount in each fraction).

Fraction	Total PG/106 cells		Non-HSPG/106 cells		HSPG/106 cells
	CPM	μg	CPM	μg	CPM	μg
Cytoplasmic	87,318 ± 1,323	0.517	26,800 ± 2,723	0.159	60,518	0.358
Nuclear	14,044 ± 1,025	0.083	4,185 ± 68	0.025	9,859	0.058