Figure 1. PRMT5 interacts with FAM47E.

(A) Y2H assay was performed to study the interaction between PRMT5 with FAM47E. The pGBKT7-PRMT5 and PGADT7-FAM47E constructs were allowed to interact with each other or corresponding vector controls. The positive interaction was assessed by the investigating the expression of reporter genes. SD−Trp/−Leu denotes the synthetically defined medium which lacks tryptophan and leucine, SD−Trp/−Leu/+Aba/+X-α-Gal denotes synthetically defined medium which lacks tryptophan and leucine but contains Aureobasidin A and X-α-Gal and SD−Trp/−Leu/−His/−Ade/+Aba/+X-α-Gal denotes synthetically defined medium which lacks tryptophan, leucine, histidine, and adenine but contains Aureobasidin A and X-α-Gal. (B) HEK293 cells were co-transfected with Myc-PRMT5 and GFP or GFP-FAM47E constructs. Co-immunoprecipitation (Co-IP) was performed using GFP-Trap and the bound fractions were probed with Myc antibody. About 0.5% of the whole cell lysates which were used in Co-IP were probed with Myc antibody or GFP antibody. (C) HEK293 cells were co-transfected with HA-FAM47E and GFP or GFP-PRMT5 constructs. Co-IP was performed using GFP-Trap and the bound fractions were probed with HA antibody. About 1% of the whole cell lysates which were used in Co-IP were probed with HA antibody or GFP antibody. (D) Endogenous PRMT5 interacts with GFP tagged FAM47E. HEK293 cells were transfected with GFP or GFP-FAM47E constructs. Co-IP was performed using GFP-Trap and the bound fractions were probed with PRMT5 antibody. About 2% of the whole cell lysates which were used in immunoprecipitation were probed with PRMT5 antibody or GFP antibody. (E) PRMT5 interacts with FAM47E directly. GST pull-down assay was carried out using recombinant GST-FAM47E and His-RMT5 proteins. The bound fractions were probed with His antibody. About 4% of His-PRMT5 (middle blot) and 2% of GST-FAM47E (lower blot), which were used in the pull-down assay, were resolved in SDS–PAGE and stained with coomassie blue dye. (F) PRMT5 interacts with FAM47E at their endogenous levels in HEK293 cells. The cell lysates were prepared from HEK293 cells and immunoprecipitations were performed using rabbit IgG or FAM47E antibody and the bound fractions were probed with PRMT5 antibody. About 2% of the whole cell lysates which were used in immunoprecipitation were probed with PRMT5 antibody or FAM47E antibody. (G) The cell lysates were prepared from HEK293 cells and immunoprecipitations were performed using rabbit IgG or PRMT5 antibody and the bound fractions were probed with FAM47E antibody. About 0.1% of the whole cell lysates which were used in immunoprecipitation were probed with PRMT5 antibody or FAM47E antibody.

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