Figure 5.

Reduced expression level of the P477L variant MUNC18-2 protein. (A) Western blot analysis of GFP–MUNC18-2 after transient transfection of HEK293T cells using the anti-GFP antibody. (B) Fluorescence-activated cell sorter analysis of GFP–MUNC18-2 expression in WT and Munc18-2 KO organoids. Continuous blasticidin antibiotic selection was performed to ensure homogenous expression. Histogram plot of GFP intensity shows reduced expression of the P477L point mutant protein compared with the WT. (C) Immunohistologic detection of GFP–MUNC18-2 expression in WT and Munc18-2 KO organoids using the anti-GFP antibody on paraffin sections. Specific apical brush-border signals could be detected (white arrows), but a more diffuse signal was detected with the point mutant protein in Munc18-2 KO organoids. Magnified images (boxes) are shown in the bottom rows. Scale bars: 50 μm. Staining was confirmed in 2 independent experiments. con, negative control; mut, mutant.