Fig. 3
In planta accumulation of VLRM1 is higher if the protein goes through the secretory pathway. a Western blot of cytoplasmic and apoplastic VLRBs fused at their C-terminus with three HA tags detected with α-HA antibodies. Eight µg of total protein were loaded per well. Expression of three different high-affinity HopM1-specific VLRBs (done in duplicate) with slightly different amino acid sequences (see Fig. 2c) was performed for the cytoplasmic (VLRM1-HA3) and apoplastic (SP-VLRM1-HA3) versions of the HopM1-specific VLRB. A Ponceau S staining of the membrane is shown below the blot to confirm similar sample loading of the gel. Accumulation of only SP-VLRM1 was observed. SP signal peptide. b Western blot of cytoplasmic VLRB fused at its C-terminus with YFP detected with α-GFP antibodies. Twenty-nine µg of total protein were loaded per sample. Expression of three different high-affinity HopM1-specific VLRBs with slightly different amino acid sequences was performed. YFP and HopK1-YFP (K) were used as positive controls, while an A. tumefaciens strain (At) devoid of any plant expression vectors was used as a negative control. The asterisk represents the position of a non-specific band. A Ponceau S staining of the membrane is shown below the blot to confirm similar sample loading of the gel. c Western blot of cytoplasmic VLRB fused at its C-terminus with syntaxin SYP61 (At1g28490) and three HA tags detected with α-HA antibodies. Twenty-three µg of total protein were loaded per well. Proteins were extracted from four T1 transgenic A. thaliana Col-0 plants and an untransformed Col-0 plant. Asterisks represent the position of non-specific bands. A Ponceau S staining of the membrane is shown below the blot to confirm similar sample loading