Figure 1: Quantitative real-time PCR analysis of the GalNAc4S-6ST transcript and profile of sulfation pattern of CS in the LLC-4S6ST-shRNA cells. (a) Total RNA was extracted from the control- (mock) or GalNAc4S-6ST-shRNA/LLC cells, and cDNA was synthesized by reverse transcriptase. Real-time PCR was conducted using the cDNAs, Taq polymerase, and SYBER Green. The expression of GalNAc4S-6ST was individually normalized to that of G3pdh. The assay was performed at least twice in triplicate, and representative results are shown. Values represent the mean ± SD. *; ** versus control by one-way ANOVA with Dunnett’s adjustment. (b, c) Anion-exchange HPLC of disaccharides obtained from the digests of CS derived from control- and GalNAc4S-6ST-shRNA/LLC cells with a mixture of chondroitinases ABC and AC-II. The GAG-peptide preparations from LLC cells, which stably express control-shRNA (b) or 4S6ST-shRNA (c), were individually digested with a mixture of chondroitinases ABC and AC-II. Each digest was labeled with a fluorophore 2AB as detailed in Section 2 and analyzed by anion-exchange HPLC on an amine-bound silica PA03 column using a linear gradient of NaH2PO4 as indicated by the dashed lines. The eluate was monitored by fluorescence intensity with the excitation and emission wavelengths of 330 and 420 nm, respectively. The insets show magnified chromatograms (10-fold) around the elution position of ΔE units. The positions of the 2AB-derivatized authentic disaccharides are indicated by numbered arrows: 1: ΔO, ΔHexUA-GalNAc; 2: ΔC, ΔHexUA-GalNAc(6-O-sulfate); 3: ΔA, ΔHexUA-GalNAc(4-O-sulfate); 4: ΔD, ΔHexUA(2-O-sulfate)-GalNAc(6-O-sulfate); 5: ΔB, ΔHexUA(2-O-sulfate)-GalNAc(4-O-sulfate); 6: ΔE, ΔHexUA-GalNAc(4-O-, 6-O-disulfate); 7: ΔT, ΔHexUA(2-O-sulfate)-GalNAc(4-O-, 6-O-disulfate).