GST pulldown assay in the presence and absence of Rose Bengal. −RB: Absence of Rose Bengal; +RB: presence of Rose Bengal; GST: glutathione S-transferase. (a) Interaction between SecA and AprE, Gst-SecA: recombinant protein expressed in Gst fused in the frame with the SecA protein, and AprE-His: AprE encoding for subtilisin fused in the frame in the C-terminal region with the 6-His-tag. SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis, α-His: immunodetection using an anti-His, α-Gst: immunodetection using an anti-Gst. (b) Interaction between SecA and AmyE, AmyE-His: AmyE encoding amylase fused in the frame in the C-terminal region with the His-tag. (c) Interaction between SecA and TasA, TasA-His: TasA encoding for the biofilm component in the frame in the C-terminal region with the His-tag. (d) Interaction between Gst-SecA and TapA, TapA-His: TapA encoding for major biofilm matrix components in the frame in the C-terminal region with the His-tag. (e) Interaction between TapA and TasA in the presence or absence of Rose Bengal. Immunodetection using an anti-TasA, anti-His, and anti-Gst. (f) Extracellular protein from the culture supernatant. 1: B. subtilis: 2 and 3: B. subtilis/pKIKO9: strain expressing AprE-His in the presence or absence of Rose Bengal, 4 and 5: B. subtilis/pKIK10: strain that expresses AmyE-His in the absence or presence of Rose Bengal, and 6 and 7: B. subtilis/pKIKO15: strain that expresses Tas-His in the presence or absence of Rose Bengal.