Figure 4. Binding of MSA2-LysM_AcmA and MSA2-LysM_AcmD to L. lactis cells.

a. Anti-MSA2 antibody-treated Western blot showing binding of MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 to non-treated (lanes 1, 2 and 3) and TCA-treated (lanes 4, 5 and 6) L. lactis cells, respectively. b. Western blot treated with anti-MSA2 antibody. Lanes 1 and 3 indicate MSA2-LysM_AcmD bound to TCA-treated L. lactis cells at pH 6.2 and 3.2, respectively. The unbound protein at pH 6.2 and 3.2 is shown in lanes 2 and 4, respectively. Lane 5, positive control: MSA2-LysM_AcmA bound to L. lactis at pH 6.2. Arrow points out Pro-MSA2-LysM_AcmD and * indicates mature MSA2-LysM_AcmD. The percentage of both MSA2-LysM variants bound to L. lactis cells at pH 6.2 and 3.2 were semi-quantified (as the number of pixels present per mm² of both bands in lanes 1 or 3) divided by the total signal of the cell and supernatant fractions (number of pixels per mm² of all bands in lanes 1+2 or 3+4, respectively) is shown at the bottom of lanes 1 and 3. c. Transmission electron microscopic images of L. lactis incubated with MSA2 or MSA2 fusion proteins and subsequently incubated with rabbit anti-MSA2 antibodies and finally decorated with goat anti-rabbit IgG gold marker. Picture 1: non-treated L. lactis cells incubated with MSA2-LysM_AcmA. Pictures 2, 3 and 4 show TCA-treated L. lactis cells incubated with MSA2-LysM_AcmA, MSA2-LysM_AcmD and MSA2 proteins, respectively. Arrows indicate immunogold particles detected on the cells.