The primer sequences utilized for different site-directed mutagenesis are explained in Table 1. demonstrate a novel PTM and PAF1/PD2 conversation through SUMOylation, and inhibiting the SUMOylation of PAF1/PD2 enhance the therapeutic efficacy for PDAC. test. Data offered as mean SD. 0.05; *0.05; n.s, nonsignificant. (E) Representative images showing the PAF1/PD2 and YH2AX expression. (F to G) Immunohistochemistry analysis with anti-PAF1/PD2 antibody was performed in radiation-treated or nonradiated human PDAC samples. (F) Representative images show the PAF1/PD2 staining in indicated samples. (G) Box plot representing the composite score for PAF1/PD2 staining. The number of samples is usually = 6 TG003 for radiated and nonradiated samples. 0.05. I. Western blot analysis showing the protein expression of PAF1/PD2 in control and in radiation treated SW1990 and CD18/HPAF cells. -Actin was used Oaz1 as the loading control. Radiation induces apoptosis in malignancy cells by DNA damage, which triggers DNA damage response and genome instability. To investigate whether loss of TG003 PAF1/PD2 affects the DNA damage induced by radiation, we examined TG003 the expression of YH2AX (a molecular marker for double-strand breaks) (23) in cells with PAF1/PD2 knockdown. The PAF1/PD2 knockdown SW1990 cells TG003 showed a slight increase in YH2AX levels (not significant) induced by radiation. However, the YH2AX significantly increased by radiation in PAF1/PD2 knockdown CD18/HPAF cells (Fig. 1D and ?andE).E). These results indicate that this PAF1/PD2 protein may impact the DNA damage response machinery in malignancy cells in response to radiation. Correspondingly, we further analyzed PAF1/PD2 expression in radiation-treated and nontreated human PDAC tissue samples. Compared with non-radiation-treated samples, we observed that PAF1/PD2 expression is usually upregulated, although nonsignificantly in radiation treated PDAC samples ((Fig. 3A). Furthermore, the JASSA analysis demonstrated a high cutoff TG003 score for K106, K150, and K154 (Fig. 3B). We generated PAF1/PD2 K47R, K106R, K133R, K150R, K154R, and K150/154R mutants (lysine residues were mutated to arginine (R)) and transfected them and the wild-type Flag-tagged PAF1/PD2 together with HA-UBC9 and HA-SUMO1 in HEK293T cells. Site-directed mutation at K106 and K150/154R sites of PAF1/PD2 largely reduced the SUMO modification on PAF1/PD2 (Fig. 3C and ?andD).D). Moreover, a double mutation at sites K150 and K154 to arginine (K150/154R) significantly decreased the PAF1/PD2 SUMOylation compared with wild-type PAF1/PD2 or PAF1/PD2 with K106R mutation (Fig. 3E). In our present study, we focused on the activity of K150 and K154 (hereafter PAF1/PD2 K150/154R is usually annotated as PAF1/PD2 KR mut.), because these sites showed the most significant reduction in SUMOylation compared to the WT (Fig. 3D) and these lysine residues (K150 and K154) are also more conserved than K106 (Fig. 3A). Open in a separate windows FIG 3 Identification of candidate SUMO sites on PAF1/PD2. (A) Alignment of PAF1/PD2 made up of the putative SUMOylation site; K47, K106, K133, K150, and K154 from multiple species. The putative SUMOylation site is usually highlighted in reddish. (B) JAASA software prediction of candidate SUMO acceptors on PAF1/PD2. The site was highlighted in blue. (C to D) HA-SUMO1 plasmids were cotransfected with Flag-PAF1/PD2 WT or Flag-PAF1/PD2 lysine single-mutant plasmids to HEK293T cells. (C) Immunoprecipitation with anti-Flag and immunoblotting with anti-HA antibody represents the conversation between the two proteins. The whole-cell lysate (WCL) was immunoblotted with anti-Flag, anti-HA, and -actin antibodies. (D) The bar graph showing the quantified result of the fold switch of SUMO-PAF1/PD2 (represented by HA in the IP data) to total PAF1/PD2 (represented by Flag in the IP data). As the first two groups do not involve the transfection with Flag-PAF1/PD2 and did not show any band for Flag, it was not included in the graph. Values are offered as mean SE, PLA assay in SW1990 cells. We observed that treatment with radiation significantly increased the localization of PAF1/PD2 with PML as observed through quantity of PLA foci, an observation that has by no means been resolved before (Fig. 4A and ?andB).B). In addition, our analysis revealed that radiation enhanced the colocalization of PAF1/PD2, SUMO1, and PML in.