Journal: Journal of Virology
Article Title: Minichromosome Maintenance Proteins Cooperate with LANA during the G 1 /S Phase of the Cell Cycle To Support Viral DNA Replication
doi: 10.1128/JVI.02256-18
Figure Lengend Snippet: LANA and MCMs are part of the replication complex. (A) Schematic of two-step iPOND performed on KSHV-positive cells. Approximately 100 million KSHV-positive cells were labeled with 5-ethynyl-2′deoxyuridine (EdU) for 30 min, harvested, and washed with 1× PBS (1). Protein and DNA were cross-linked using 1% formaldehyde for 20 min and quenched using 125 mM glycine (2). The cells were permeabilized with 0.25% Triton X-100 in PBS for 30 min at room temperature (3), and nuclei were isolated following centrifugation (4). Click chemistry was performed on the nuclei to conjugate biotin-azide to EdU, and DMSO was used as a negative control (5). The chromatin was sheared using sonication to generate fragments of 100 to 300 bp (6). Protein-DNA complex bound to LANA was captured using monoclonal LANA antibody (7). Protein A/G beads were used to capture antigen-antibody complexes, which were eluted from the beads using peptide specific for LANA (8). Streptavidin beads were used to capture EdU-labeled (replicated) DNA-protein complexes (9). Proteins bound to streptavidin beads were eluted by boiling the beads at 95°C and detected using specific antibodies (10). (B) Immunoblot of LANA, MCM6, and PCNA obtained through two-step iPOND using respective antibodies from KSHV-positive BCBL-1 and BrK.219 cells. A total of 100 million cells were labeled with EdU and permeabilized, and a click reaction was performed using biotin-azide and DMSO. The cells were lysed and sonicated, and proteins were pulled down using LANA antibody. The proteins associated with DNA were pulled down using streptavidin beads and eluted by boiling in the loading buffer.
Article Snippet: The following commercial antibodies were used for this study: rat anti-LANA (Advanced Biotechnologies, Inc.); mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (US Biological); mouse anti-Flag M2 (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-Myc 9E10 (Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-MCM2, -MCM5, -MCM10, and -PCNA (Santa Cruz Biotechnology); mouse monoclonal anti-MCM3, -MCM4, and -MCM7 (Santa Cruz Biotechnology).
Techniques: Labeling, Isolation, Centrifugation, Negative Control, Sonication, Western Blot
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