Extended Data Fig. 4: Immunoblot detection of AvrE and DspE proteins and elution profile of MBP-DspE and MBP-DspEΔβ-barrel proteins.
From: Bacterial pathogens deliver water- and solute-permeable channels to plant cells
a, Oocyte surface biotinylation assay of DspE. Two nanograms of wild-type or mutant dspE cRNA was injected into oocytes, which were incubated in ND96 media at 18 °C for 15 h before being subjected to surface protein biotinylation assay (see Methods). Anti-β-Actin antibody detect β-Actin, a cytoplasmic protein serving as a negative control. b,c,e,f, Detection of AvrE and DspE proteins expressed in oocytes. Oocytes injected with 1 ng (unless otherwise noted in the gel lane label) avrE, dspE or mutant dspE cRNA or H2O (control) were incubated in ND96 media at 18 °C for 19 h before being subjected to SDS-PAGE and immunoblot analysis. In c, oocytes were incubated in the absence or presence of 5 or 10 mM PAMAM G1. d, Detection of DspE proteins expressed in tobacco leaves. 1 × 108 CFU/mL of Agrobacterium tumefaciens GV3101 containing pER8 empty vector, pER-dspE or pER-dspE mutants were syringe-inoculated into leaves of Nicotiana benthamiana, kept at 22 °C for 24 h before leaves were painted with 90 μM estradiol to induce protein expression for 8 h and then subjected to SDS-PAGE and immunoblotting. Asterisks in the pER8 and DspEWT lanes indicate a faint nonspecific protein band. Because active DspE expressed much more poorly in plant cells than mutant DspE proteins, mutant DspE samples were diluted 20 times with 2×SDS sample buffer (See Supplementary Fig. 1d). Experiments were independently performed two times. See Supplementary Fig. 1 for image cropping. g,h, Purified WT MBP-DspE (g) or MBP-DspEΔβ-barrel (h) was eluted on a Suprose 6 increase 10/300 GL column and was analyzed on a SDS-PAGE gel. FPLC traces and gel images in g and h are representative of 3 experimental replicates. See Supplementary Information Fig. 1 for image cropping.
