Anti r glcnac and anti his tag monoclonal antibodies

In vitro glycosylation assays were conducted as previously described [1 (link)]. Enzymes (200 nM of NleB1, NleB, or SseK1) were incubated in 50 mM Tris-HCl buffer pH 7.4, 1 mM UDP-GlcNAc, 10 mM MnCl₂, and 1 mM DTT with 1 mM GAPDH in the presence or absence of serial dilutions of avasimibe. After a 2 h incubation at room temperature, samples were blotted with anti-R-GlcNAc and anti-His tag monoclonal antibodies (Abcam, Cambridge, MA, USA). LI-COR Image Studio software (LI-COR Biosciences, Lincoln, NY, USA) was used to measure signal intensities, and inhibition was estimated by measuring the relative reduction in substrate glycosylation.

In vitro glycosylation assays were performed as described previously [24 (link)]. Enzymes (200 nM of NleB1, NleB, SseK1, or SseK2) were incubated with 1 µM of GAPDH or FADD +/− serial dilutions of YM155 in 50 mM Tris-HCl buffer pH 7.4, 1 mM UDP-GlcNAc, 10 mM MnCl₂, and 1 mM DTT. After 2 h incubation at RT, samples were subjected to western blotting using anti-R-GlcNAc and anti-His tag monoclonal antibodies (Abcam, Cambridge, MA, USA). Signal intensities were quantified using LI-COR Image Studio software (LI-COR Biosciences, Lincoln, NY, USA), and inhibition was calculated by quantifying the relative reduction in substrate glycosylation.

In vitro glycosylation assays were conducted as previously described^{7 (link)}. 200 nM SseK1 or SseK1 HEN were incubated in 50 mM Tris–HCl buffer pH 7.4, 1 mM UDP-GlcNAc, 10 mM MnCl₂, and 1 mM DTT with 1 mM OmpR. After a 2-h incubation period at room temperature, samples were blotted with anti-R-GlcNAc and anti-His tag monoclonal antibodies (Abcam, Cambridge, MA, USA). Western blot images were captured in a LI-COR (LI-COR Biosciences, Lincoln, NY, USA) imager.