SULF expression in corneal epithelial cells.
A: RT-PCR analysis for SULF expression was performed on cDNA prepared from THCE, MCE and 293T cells transfected with pcDNA Sulf1 (S1) or pcDNA Sulf2 (S2) as positive controls. First-strand cDNA synthesis was also performed with reverse transcriptase omitted as a control for genomic DNA contamination (RT-). B: Detergent lysates of human primary corneal epithelial cells (PCE), THCE, MCE and pcDNA SULF1 (S1) or pcDNA SULF2-transfected 293T (S2) cells were subjected to Western blot analysis with 1A4 (SULF1) and R2.1/2.3 (SULF2). SULF1 was detected in MCE cells by the presence of its C-terminal subunit (∼50 kDa), while SULF2 was absent. SULF2 was detected in THCE and PCE cells (as the 125 kDa proprotein). C: SULF2 in the indicated THCE cells. Western blotting for SULF2 (R2.1/R2.3) was performed on conditioned media (CM) and detergent lysates from these cells. Density of each band was measured with ImageJ, values were normalized to β-actin of the Cnt shRNA-treated cells, and the percent SULF2 expression (shown at the bottom of the respective lanes) was calculated relative to the expression of the protein in Cnt shRNA cells. shRNA1 and shRNA2 reduced SULF2 expression by ∼83% and ∼98% respectively. D: and E: Effects of SULF2 knockdown on the cell-surface sulfation was determined by staining with RB4CD12. D: Immunofluorescent staining of RB4CD12 or control MPB49 antibody in THCE control- and SULF2-knockdown cells. E: Flow cytometry analysis of RB4CD12 or control MPB49 staining in THCE control- and SULF2-knockdown cells. SULF2 knockdown increased the level of RB4CD12 epitope on the cell surface by 72±2% (mean ± SEM, n = 3 experiments). A representative result is shown.