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. 2020 Mar 11;9(6):e010240. doi: 10.1161/JAHA.118.010240

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© 2020 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Both mPinX1 and mPinX1t proteins bound cardiac transcription factor mRNAs, while only mPinX1t protein but not mPinX1 protein‐bound Nucleoporin 133 (Nup133). A through D, RNA immunoprecipitation of HEK293FT cells overexpressed with (A and C) myc‐mPinX1 or (B and D) myc‐mPinX1t and (A and B) Gata4 or (C and D) Tbx5. Anti‐myc was used to perform the immunoprecipitation. IgG2a was used in isotype control experiment. The presence of (A and B) Gata4 mRNA or (C and D) Tbx5 mRNA in the immunoprecipitant was quantitated by subsequent quantitative polymerase chain reaction. The results showed that both mPinX1 and mPinX1t proteins could bind to Gata4 mRNA and Tbx5 mRNA. Data were presented as mean±SEM (n=3; where n represents independent RNA immunoprecipitation experiments). *P<0.05 vs control. E, Coimmunoprecipitation assay of HEK293FT cells overexpressed with myc‐mPinX1t (left panel) or myc‐mPinX1 and HA‐Nup133 (right panel). (Left panel) In the immunoprecipitant of anti‐myc (which contained myc‐mPinX1t), HA‐Nup133 was detected. No HA‐Nup133 was detected in the isotype control group. (Right panel) In the immunoprecipitant of anti‐myc (which contained myc‐mPinX1), HA‐Nup133 was not enhanced when compared with isotype control group. Another control experiment was also performed in which myc only and HA‐Nup133 were overexpressed in HE293FT cells. Using anti‐myc for immunoprecipitation, as expected, no HA‐Nup133 was detected in the immunoprecipitant. F, Coimmunoprecipitation assay of HEK293FT cells overexpressed with myc‐mPinX1t. As shown in the input lane, HEK293FT expressed endogenous Nup133. In the immunoprecipitant of anti‐myc (which contained myc‐mPinX1t), endogenous Nup133 was detected. No Nup133 was detected in the isotype control group. DAPI indicates 4′,6‐diamidino‐2‐phenylindole.