Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells.

<p><b>A</b>. HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total ERK1/2 expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. <b>B</b>. HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. <b>C</b>. NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.