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. 2018 Jun 25;35(2):171–176. doi: 10.5511/plantbiotechnology.18.0412a

Figure 1. Overexpression of MAP3K17 and its kinase-inactive mutant in Arabidopsis. (A) Schematic representation of the constructs for the overexpression of 3xFLAG-MAP3K17 (FL) and 3x-FLAG MAP3K17KN (K32R, KN). The 3xFLAG-tag was translationally fused to the N-terminus of the coding sequence, without an initiation codon, of MAP3K17 and MAP3K17 KN and then inserted between the CaMV 35S promoter (35SP) and the NOS terminator (NOS Ter) of the plant expression vector pBI121 (Clontech) using XbaI and SacI restriction enzyme sites. RB: right border, LB: left border, NOS Pro: NOS promoter, NPTII: neomycin phosphotransferase II gene, NOS Ter: NOS terminate, KD: kinase domain (B) Detection of the 3xFLAG-MAP3K17 and 3xFLAG-MAP3K17KN proteins. Total protein extracts from WT and each transgenic plant were resolved by SDS-PAGE. Immunoblot analysis was conducted using the anti-Flag tag antibody. Equal amounts of the samples were resolved by SDS-PAGE and stained with CBB, and the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown as the loading control for equal protein amounts in the WT and transgenic plants.

Figure 1. Overexpression of MAP3K17 and its kinase-inactive mutant in Arabidopsis. (A) Schematic representation of the constructs for the overexpression of 3xFLAG-MAP3K17 (FL) and 3x-FLAG MAP3K17KN (K32R, KN). The 3xFLAG-tag was translationally fused to the N-terminus of the coding sequence, without an initiation codon, of MAP3K17 and MAP3K17 KN and then inserted between the CaMV 35S promoter (35SP) and the NOS terminator (NOS Ter) of the plant expression vector pBI121 (Clontech) using XbaI and SacI restriction enzyme sites. RB: right border, LB: left border, NOS Pro: NOS promoter, NPTII: neomycin phosphotransferase II gene, NOS Ter: NOS terminate, KD: kinase domain (B) Detection of the 3xFLAG-MAP3K17 and 3xFLAG-MAP3K17KN proteins. Total protein extracts from WT and each transgenic plant were resolved by SDS-PAGE. Immunoblot analysis was conducted using the anti-Flag tag antibody. Equal amounts of the samples were resolved by SDS-PAGE and stained with CBB, and the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown as the loading control for equal protein amounts in the WT and transgenic plants.