Additional file 1 of Linear ubiquitination of p31comet by HOIP couples cytokine response with mitotic regulation

Additional file 1: Figure S1. APC/CCdc20 substrates are accumulated in HOIP-depleted cells. A-C. Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from HEK293 (A) and HeLa (B) cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. When indicated, cells were treated with 50 ng∙ml-1 TNFα when released from nocodazole release. (C) The relative intensities of securin bands were calculated by normalizing to the Vinculin loading control and then to the time 0. D-E. IB analysis of WCL derived from HeLa cells (D). Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. When indicated, cells were treated with 40 ng∙ml-1 IL-1β when released from nocodazole release. (E) The relative intensities of securin bands were calculated by normalizing to the Vinculin loading control and then to the time 0. F. IB analysis of WCL derived from HeLa cells. Cells were arrested in mitosis using 300 nM nocodazole for 16 h. Mitotic cells were collected by mechanical shake-off and released back into the cell cycle in media without nocodazole. Cells were harvested at the indicated time points after nocodazole release. When indicated, cells were treated with 5 ng∙ml-1 or 50 ng∙ml-1 TNFα when released from nocodazole release. G-I. IB analysis of WCL derived from HEK293 (G), DLD1 (H), and U2-OS (I) cells transduced with shScr (as the negative control) or the indicated lentiviral shHOIP constructs. The transduced cells were selected with 1 g∙ml-1 puromycin for 72 h before harvest. J. IB analysis of WCL derived from HCT116 cells transduced with sgGFP (as the negative control) or sgHOIP lentiviral constructs. The cells were treated with 50 ng∙ml-1 TNFα for 16 h before harvest. K-L. IB analysis of WCL derived from DLD1 (K) and HEK293 (L) cells transduced with shScr (as the negative control) or the indicated lentiviral shHOIL-1L constructs. The transduced cells were selected with 1 g∙ml-1 puromycin for 72 h before harvest. M-N. IB analysis of WCL derived from DLD1 (M) and HEK293 (N) cells transduced with shScr (as the negative control) or the indicated lentiviral shSHARPIN constructs. The transduced cells were selected with 1 g∙ml-1 puromycin for 72 h before harvest.