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. 2016 Jan 14;113(5):1273–1278. doi: 10.1073/pnas.1500992113

Fig. 2.

Fig. 2.

UPAT is associated with UHRF1 in colon cancer cells. (A) Nuclear extracts from HCT116 cells were incubated with biotinylated sense, antisense UPAT (Left), or ASBEL (Right) generated in vitro, and proteins were precipitated with streptavidin beads and subjected to immunoblotting analysis with anti-UHRF1 or anti-Actin antibody. AS-UPAT, in vitro-transcribed antisense UPAT. Actin was used as a negative control. (B) Lysates from HCT116 cells were subjected to immunoprecipitation with anti-UHRF1 antibody or anti-mouse IgG antibody followed by qRT-PCR analysis to detect UPAT mRNA. GAPDH mRNA, U1 small nuclear RNA, ASBEL, and UCA1 were used as negative controls. Results are expressed as the mean ± SEM (n = 3). (C) Lysates from HCT116 cells transfected with sense (UPAT) or antisense (AS-UPAT) UPAT and HA-UHRF1 were subjected to immunoprecipitation with anti-HA antibody followed by RT-PCR analysis to detect UPAT mRNA. AS-UPAT and GAPDH were used as negative controls. Results are expressed as the mean ± SEM (n = 3). (D) Schematic representation of the UHRF1 protein (Upper) and UPAT (Lower). Mutants used in RIP (Fig. 2 E–H), immunoprecipitation (Fig. 3E and Fig. S4H), and ubiquitination (Fig. 4A and Fig. S5B) assays are also shown. (E–G) Lysates from HCT116 cells transfected with UPAT along with wild-type, mutant HA-UHRF1 (E), or mutant Flag-UHRF1 (F and G) were subjected to immunoprecipitation with anti-HA (E) or anti-Flag (F and G) antibody followed by RT-PCR analysis to detect UPAT and U1 small nuclear RNA. Results are expressed as the mean ± SEM (n = 3). See also Fig. S3E. (H) Lysates from HCT116 cells transfected with wild-type or mutant UPAT and HA-UHRF1 were subjected to immunoprecipitation with anti-HA antibody followed by RT-PCR analysis to detect UPAT mRNA. Results are expressed as the mean ± SEM (n = 3). (I) qRT-PCR analysis of UHRF1, SCD1, and SPRY4 expression in HCT116 cells transfected with siRNA targeting UHRF1. Results are expressed as the mean ± SEM (n = 3). (J) Viability of HCT116 and CCSC#P cells transfected with siRNA targeting UHRF1 was assessed by Cell Titer-Glo assays. Results are expressed as the mean ± SEM (n = 3). *P < 0.05.