SMIM45-107aa interacted with MTDH and inhibited MTDH ubiquitination. A Schematic diagram of the detection process for SMIM45-107aa interaction proteins. B IP assays were performed using anti-Flag agarose beads in SK-Hep1 andor QGY-7701 cells overexpressing SMIM45-107aa. Mut: SMIM45-107aa-mutation; OE: SMIM45-107aa-overexpression. C The immune precipitates were detected using SDS-PAGE. Mut 1: SMIM45-107aa-mutation in SK-Hep1 cells; OE 1: SMIM45-107aa-overexpression in SK-Hep1 cells; Mut 2: SMIM45-107aa-mutation in QGY-7701 cells; OE 2: SMIM45-107aa-overexpression in QGY-7701 cells. D Veen diagram of MS data in SK-Hep1 and QGY-7701 to detect the interaction proteins with SMIM45-107aa. E–F Co-IP was performed after overexpressing SMIM45-107aa in SK-Hep1 andor QGY-7701 cells. G Co-IP was performed after overexpressing SMIM45-107aa in HEK-293T cells. Cellular lysates were treated with or without 30 µg/ml RNase A for 30 min, followed by Co-IP using anti-MTDH antibody or anti-Flag resin. H–I Immunofluorescence assays were performed to visualize the co-localization of SMIM45-107aa and MTDH in SK-Hep1 and QGY-7701 cells. Scale bars, 75 μm. (J–K) SMIM45-107aa-Flag plasmid J and MTDH-Myc plasmid K were transfected into HEK-293T cells respectively. Expression was analyzed by RT-PCR and Western blot analysis. L SMIM45-107aa was knocked down by siRNA in QGY-7701 cell line. Expression levels were detected by RT-PCR and Western blot. (M, N) Co-IP assays showing the effect of SMIM45-107aa overexpression on MTDH polyubiquitination in HEK-293T cells and QGY-7701
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