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. 2016 Feb 5;2(2):e1501257. doi: 10.1126/sciadv.1501257

Fig. 1. PAD4-mediated citrullination of E2F-1 augments its transcriptional activity.

Fig. 1

(A) In vitro citrullination of GST (glutathione S-transferase)–E2F-1by Flag-PAD4, where recombinant GST–E2F-1 (1 μg) was incubated with Flag-PAD4 (1 μg). Lanes 1 to 7 were in the presence of CaCl2, and lane 8 in the absence of CaCl2. The arrow points to citrullinated GST-E2F-1. AMC, anti-modified citrulline (Millipore); IB, immunoblot. (B) Citrullination of E2F-1 in U2OS cells, transfected with HA (hemagglutinin)–PAD4 (2 μg). The lysate was incubated with 4 mM CaCl2 and 2 mM dithiothreitol (DTT). E2F-1 (C20) and rabbit immunoglobulin G (IgG) antibodies were used for immunoprecipitation (IP). (C) Citrullunation of N-terminal domain of E2F-1 in U2OS cells, transfected with Flag–E2F-1 (1 μg) and HA-PAD4 (2 μg) and treated with A23187 (5 μM, 30 min). Flag-agarose beads were used for immunoprecipitation. ΔC E2F-1: 1 to 194, ΔN E2F-1: 194 to 437. (D) Interaction between HA–E2F-1 and Flag-PAD4 in Flag-PAD4.pTRE and pTRE empty vector cell lines transfected with pcDNA or HA–E2F-1 (1 μg). Flag-agarose beads were used for immunoprecipitation. (E) Relative luciferase reporter comparing HA–E2F-1 wild-type (WT) and R4K activity in U2OS cells transfected with HA–E2F-1 WT or R4K mutant (R109K/R111K/R113K/R127K) (100 ng), Flag-PAD4 (300 ng), p73 luciferase reporter (100 ng), and β-gal (150 ng) plasmid. The luciferase reporter signal was normalized to β-gal reading. (F) Accompanying immunoblot ± SD. *P < 0.05. (G) Quantitative polymerase chain reaction (qPCR) analysis comparing Flag ΔC E2F-1 WT and R4K relative promoter occupancy in U2OS cells transfected with Flag ΔC E2F-1 WT or R4K mutant (R109/R111/R113/R127) (1 μg) and HA-PAD4 (2 μg). Flag antibody was used for ChIP. (H) Accompanying immunoblot ± SD. *P < 0.05. (I) ChIP analysis in doxycycline-inducible Flag-PAD4.pTRE cells {treated with or without doxycycline (1 μg/ml) [P4(+) and P4(−), respectively]}, measuring PAD4 promoter occupancy in nontargeting (NT) versus E2F-1 siRNA (30 nM)–transfected cells, and presented as visualized on ethidium bromide–stained gel. NS, nonspecific. (J) Accompanying immunoblot.