Fig 3. Protein solubility, polysaccharide binding and cell wall protection assays.
A. Polyacrylamide gel analysis of protein solubility. The protein samples were centrifuged at a maximum speed for 5 min and aliquots of each sample (10 μg each) were analyzed on a 12% SDS-PAGE gel. M, protein marker. B. Protein binding assay. After incubation of WT Blys2, truncated forms of Blys2 and Blys5 proteins with various polysaccharides, the supernatant and polysaccharide precipitated samples were run through a 12% SDS-PAGE gel and stained with the Coomassie brilliant blue to examine the protein-binding features. Ch, chitin extracted from fungal walls; Chb, chitin beads; Chs, chitosan; Cel, cellulose. The truncated forms of Blys2 are indicated as, e.g., Blys2D1-2 for the truncated Blys2 only containing the first two LysM domains and Blys2D1-4 for the truncated Blys2 only containing the 1–4 LysM domains etc. C. Cell wall protection from chitinase hydrolysis. The germlings were pre-incubated with either Blys2 or Blys5 for 2 hrs before the treatment with a chitinase cocktail for 2 hrs. The release rate of protoplast was quantified and compared. The WT and WT::Slp1 germlings without the pre-incubation with either protein were treated with chitinase cocktails as controls. ***, P < 0.0001.