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. 2017 Jul 1;26(13):973–985. doi: 10.1089/scd.2016.0331

FIG. 7.

FIG. 7.

HIF-1α and SUMO1 increase nuclear translocation of N1ICD in HEK293T cells. (A) Representative western blot and quantification of N1ICD by densitometry in transiently transfected HEK293T cells (black bars for HEK293T and gray bars for N1ICD-293T cells). Data represent mean ± SEM of three independent experiments. (B) RT-qPCR analysis of Notch downstream genes Hes1, Hey1, and Hey2 in HEK293T (black bars) and N1ICD-293T cells (gray bars). Data represent mean ± SEM of three independent experiments. (C) Representative western blot and quantification by densitometry of HIF-1α to assess hypoxia induction and N1ICD in HEK293T and N1ICD-293T cells treated or not with AA. (D) RT-qPCR analysis of Notch downstream genes Hes1, Hey1, and Hey2 in HEK293T and N1ICD-293T cultured in the presence (+) or absence (−) of hypoxia (Hx) and AA. Data represent mean ± SEM of three independent experiments. (E) Immunoprecipitation of N1ICD. HEK293T transduced with an N1ICD lentiviral vector (N1ICD +) cultured under normoxic or hypoxic conditions (Hx +/−) and treated or not with AA (+/−) were lysed and cell extracts were immunoprecipitated with an anti-N1ICD antibody, resolved by SDS-PAGE, and immunoblotted with anti-SUMO1 antibody. Molecular weights of protein markers are indicated at the left side of the panel in kDa. Data represent mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01, ***P < 0.001.