ion of GalNAc-b3-GlcNAc-b4-Man-a-MU A large scale reaction was carried out using GlcNAc-b4-Man-a-MU that had been produced by Sussex Research Labs to make the final product GGM-MU. B3GALNT2dTM containing a 6xHis Tag was bound to metal affinity resin and added to 9 mM UDP-GalNAc and 9 mM GM-MU in 100 mM MES, pH 6.0, 10 mM MgCl2, and 10 mM MnCl2 and incubated for 4872 hr at 37C with rotation. At the end of this time there was about 7080% conversion of substrate GM-MU to produce GGM-MU. The sample was then run over a C18 column with buffer A and buffer B. Using a 16% buffer B isocratic gradient and a flow rate of 3 ml/min, the product GGM-MU gave a signal at around 27 min by fluorescence. This peak was collected, lyophilized, and brought up in ultra-pure water and quantitated using fluorescence and GlcA-MU as a standard. In vitro kinase assay POMK kinase assay was performed as described in the Fam20B kinase assay. Reactions were carried out in 50 mM HEPES, pH 7.5, 10 mM MnCl2, 20 mM Zhu et al. eLife 2016;5:e22238. DOI: 10.7554/eLife.22238 12 of 18 Research article Biochemistry Biophysics and Structural Biology GGM-MU, 100 mM ATP and 1 mg/ml POMK for 30 min at 20C, and terminated with 20 mM EDTA and 15 mM ATP. 
The reaction TSU 68 biological activity mixtures were then loaded onto Sep-Pack C18 cartridges pre-equilibrated with 0.2 M 2SO4. Columns were washed with 2 ml of 0.2 M 2SO4 for three times, and the substrates were eluted with 1 ml methanol. Incorporated radioactivity was measured by liquid scintillation counting. Generation and characterization of HAP1 mutant cell lines HAP1 cells are a haploid human cell line with an adherent, fibroblast-like morphology, originally derived from parent cell line KBM-7. A protocol for generating HAP1 cells was previously published. HAP1 cells bearing a 10 bp deletion of exon 4 of the protein O-mannose kinase gene, generated using the CRISPR/Cas9 system, were purchased from Horizon Discovery. The identity of the cells has been authenticated by the company using the STR profiling method. Mycoplasma testing of the cells were performed on a routine basis to ensure the cells are not contaminated. POMK knockout HAP1 cells lack the single copy of the wild-type POMK allele and are therefore null at the POMK locus. The sequence of the guide RNA used is TGAGACAGCTGAAGCGTGTT. Absence of the wild-type POMK allele was confirmed by Horizon Discovery, via PCR amplification and Sanger sequencing.Adenovirus production The open reading frame of human POMK was cloned into the BamHI and XhoI sites of an expression plasmid with a C-terminal FLAG tag . E1-deficient recombinant adenoviruses were generated by the University of Iowa Viral Vector Core using the RAPAd system. Assays for replication competence of adenoviruses were performed to check for contamination. Ad-POMK-WT, Ad-POMK-A230E, Ad-POMK-Q258R, and Ad-POMK-V302D were generated by ViraQuest Inc. using the RAPAd system. Absence of the viral E1 DNA sequence was confirmed by ViraQuest Inc. after PCR amplification of the viral DNA and staining on DNA agarose gel electrophoresis. Replication competence of adenoviruses was negative as assessed by plaque forming assays in cells performed from 109 viral particles up to 14 days. Cell culture and adenovirus infection Wild-type C665 and POMK KO HAP1 cells were maintained at 37C and 5% CO2 in Iscove’s Modified Dulbecco’s PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19826115 Medium supplemented with 10% Fetal Bovine Serum and 1% penicillin-streptomycin. An average of 5.9106 POMK KO HAP1 cells were infected i
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