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. 2019 Jan 29;17(1):e2006767. doi: 10.1371/journal.pbio.2006767

Fig 4. The metazoa-specific region of MTBP is required for WT levels of DNA replication.

Fig 4

(A) Lysates of HeLa Flip-In T-Rex cells expressing siMTBP-resistant MTBP-WT-3×Flag-TEV2-GFP, MTBP-Δ516–704–3×Flag-TEV2-GFP (lacking amino acids A516–P704), MTBP-Δ516–769–3×Flag-TEV2-GFP-NLS, or MTBP-Δ516–809–3×Flag-TEV2-GFP-NLS were analysed by immunoblotting using anti-MTBP (4H9), anti-Treslin (148), and Ponceau (Pon.) staining. Error bars: SEM. (B) Cell lines of (A) were siMTBP- or siCtr-treated and analysed for replication rescue as described in Fig 3G. Averages of two (MTBP-Δ516–704–3×Flag-GFP) or four (all other lines) independent experiments are shown. (C) Whole cell lysates of HeLa Flip-In T-Rex cells expressing siMTBP-resistant MTBP-TresBD (amino acids 1–515) or MTBP-Δ516–769–3×Flag-TEV2-GFP-NLS were siCtr- or siMTBP-treated and analysed by immunoblotting as in Fig 3A. Crops of the same immunoblot exposure are shown. (D) S phase populations of cells described in (C) were analysed by BrdU-flow cytometry as in Fig 3C. AU, arbitrary units; BrdU, 5-bromodeoxyuridine; GFP, green fluorescent protein; Load., loading; MTBP, Mdm2 binding protein; Pon., Ponceau; siCtr, control RNAi; siMTBP, MTBP-RNAi; TresBD, Treslin/TICRR binding domain; WT, wild-type.