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. 2018 Oct 4;72(1):112–126.e5. doi: 10.1016/j.molcel.2018.08.043

Figure 5.

Figure 5

POLE3 and POLE4 Interact with H3-H4 In Vivo

(A) IP of endogenous POLE3 from human HeLa cells performed after CSK-Triton (0.5%) extraction on soluble and DNaseI-digested chromatin fractions. After SDS-PAGE, western blotting was performed using antibodies against the indicated proteins.

(B) FLAG IP experiments from HeLa TRex-expressing empty FLAG or FLAG-POLE4 under tetracycline-regulated promoter. Cells were induced with doxycycline for 24 hr and lysed in CSK-Triton 0.5%. FLAG IPs were performed on soluble and DNaseI-digested chromatin fractions. After SDS-PAGE and nitrocellulose transfer, membranes were incubated with antibodies against the indicated proteins.

(C) FLAG IP experiments from HeLa TRex-expressing empty FLAG, FLAG-POLE3 WT, or POLE3ΔC mutants under a tetracycline-regulated promoter. Cells induced with doxycycline for 24 hr were lysed in CSK-Triton 0.5%, and FLAG IP was performed on DNaseI-digested chromatin. After SDS-PAGE, western blotting was performed using antibodies against the indicated proteins.

(D) HA tag IP experiments from HeLa S3 cells stably expressing HA-H3.1 and HA-H3.3 from soluble and DNaseI-digested chromatin fractions. After SDS-PAGE, western blotting was performed using antibodies against HA tag, POLE3, and POLE4.

(E) FLAG IPs from HeLa TRex-expressing empty FLAG or POLE4-FLAG mutants under tetracycline-regulated promoter. Cells were induced with doxycycline for 24 hr and lysed in CSK-Triton 0.5%. FLAG IPs were performed on DNaseI-digested chromatin fractions; after SDS-PAGE and western blotting, membranes were incubated with antibodies against marks specific of newly synthesized or parental histones.

(F) Sequential IP experiments performed on empty FLAG or FLAG-POLE3 HeLa SNAP-HA-H3.1-transfected cells. After cell lysis in CSK-Triton 0.5%, chromatin was solubilized with benzonase and incubated with anti-FLAG agarose beads. After subsequent FLAG bead elution in 3xFLAG peptides (1 mg/mL concentration), HA IPs were performed, followed by SDS-PAGE and western blotting using antibodies against the indicated proteins.