DTX3L interacts with the IFNGR complex and together with ARTD9 antagonistically regulates the phosphorylation of STAT1 on Y701 in PC3 cells. (A and B) Immunoblot analyses of STAT1-signaling in PC3-siMock, PC3-siDTX3L (A), and PC3-siARTD9 (B) single knockdown cells. PC3-siMock, PC3-siDTX3L or PC3-siARTD9 single knockdown cells were treated with or without IFNγ (200 U/ml) for 2 h and then whole cell extracts separated by SDS PAGE, blotted and subsequently probed with antibodies for STAT1, pSTAT1(Y701), pSTAT1(S727) and tubulin. The immunoblots are representative of at least three independent experiments. (C and D) Quantification of pSTAT1(Y701) and pSTAT1(S727) levels shown in Figure 5A, B. pSTAT1(Y701) and pSTAT1(S727) levels were normalized to tubulin and STAT1. Values represent the mean of three independent experiments, and the error bar represents the SE. Statistical analysis was performed using the Student's t test. *P < 0.05, **P < 0.001 and ***P < 0.0001. (E and F) Co-immunoprecipitation analyses of endogenous DTX3L-IFNGR complexes in PC3 cells: Endogenous DTX3L/IFNGR complexes were co-immunoprecipitated using an anti-DTX3L antibody. Complexes were then separated on SDS PAGE, blotted and subsequently probed with antibodies against endogenous DTX3L, ARTD9, STAT1, IFNGR1, JAK1, JAK2, PTPN1 and PTPN2.