FIG 5 .

AG-PG ligase activity of ^MtbLcp1 using a radiolabeled cell-free functional assay. (A) Organic solvent-extracted fractions from ¹⁴C-labeled assay mixtures were analyzed by TLC and developed in CHCl₃-CH₂OH-H₂O-NH₄OH (65:25:3.6:0.5, vol/vol/vol/vol) on aluminum-backed silica gel plates (5735 silica gel 60F254; Merck), and products were visualized by autoradiography, exposing the TLCs to X-ray film (Kodak X-Omat). TLC is representative of three independent biological replicate experiments, and corresponding densitometric data plotted represent the mean ± standard deviation for each band. Associated P values are as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) Insoluble material from fraction 2 of the assay mixtures was hydrolyzed in 2 M TFA, reduced, and per-O-acetylated. The resulting ¹⁴C-labeled per-O-acetylated alditol acetate derivatives were analyzed by TLC using ethyl acetate-hexane (4:6, vol/vol) on aluminum-backed silica gel plates (5735 silica gel 60F254; Merck), and products were visualized by autoradiography, exposing the TLCs to X-ray film (Kodak X-Omat), and compared to known alditol-acetate standards of d-galactose (Gal), d-glucose (Glc), and d-N-acetylglucosamine (GlcNAc). TLC is representative of three independent biological replicate experiments, and corresponding densitometric data plotted represent the mean ± standard deviation for each band. Associated P values are as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.