Fig. 3. The PAF1 complex interacts with PHF6 and plays a critical role in radial migration of neurons in the cerebral cortex in vivo.

(A) Lysates of 293T cells stably expressing N-terminally tagged HA-PHF6 were immunoprecipitated with the HA beads and subjected to proteomic analyses using LC-MS/MS. Peptide spectra were analyzed and scored using the CompPASS software program. High-confidence candidate interacting proteins (HCIPs) of PHF6 are shown. Rows in pink denote components of the PAF1 complex. (B) Lysates of 293T cells expressing HA-PHF6 or HA-GFP were subjected to immunoprecipitation with the HA beads followed by immunoblotting with the HA, PAF1, LEO1, CDC73 and CTR9 antibodies. Input was also immunoblotted with the antibodies. (C) Lysates of E17 mouse cerebral cortex were immunoprecipitated with the PHF6 antibody or rabbit IgG followed by immunoblotting with the PAF1, LEO1, CDC73, CTR9 and PHF6 antibodies. Asterisk denotes a non-specific band. Rabbit IgG signal from the PHF6 immunoblot is non-specific immunoreactivity from the rabbit heavy chain. (D) Lysates of primary rat cortical neurons or mouse cerebral cortex were immunoblotted with the PAF1, LEO1, CDC73, CTR9 and ERK antibodies. (E) Lysates of mouse cortical neurons infected with lentivirus expressing PAF1 shRNAs or control shRNAs were immunoblotted with the PAF1 and 14-3-3β antibodies. (F) Lysates of Neuro2A cells infected with lentivirus expressing PAF1 shRNAs or control shRNAs were immunoblotted with the PAF1, LEO1, CDC73, CTR9 and 14-3-3β antibodies. (G) E14 embryos were electroporated with an RNAi plasmid encoding PAF1 shRNAs or control shRNAs, and analyzed as in Figure 1D. (H) Quantification of the percentage of GFP positive neurons in distinct regions of the cerebral cortex in E19 mouse embryos treated as in (G) is presented as mean + SEM. Scale bar: 50μm in (G).