Inclusion formation in Myo5b KO mice is dependent on Pacsin 2. (A) H&E micrographs of the duodenum of control, Myo5b KO, and Myo5b KO mice crossed with Pacsin 2 KO mice (double KO mice, Myo5b KO;Pacsin 2 KO). (B) Crossing of Pacsin 2 KO mice with Myo5b KO mice to achieve a double KO of Myo5b and Pacsin 2 resulted in a significant decrease in P-ERM–positive (red) inclusions, suggesting that Pacsin 2 is required for inclusion formation. Despite decreased inclusion formation in Myo5b KO;Pacsin 2 KO (double KO) mice, DDPIV (green) was still mislocalized. (C) Inclusions, as defined by subapical P-ERM immunostaining, in three 20× fields were quantified per mouse to determine the impact of loss of Pacsin 2 on inclusion formation. Quantification of inclusions in the proximal small intestine (duodenum) demonstrated that Myo5b KO;Pacsin 2 KO (double KO) mice had significantly fewer inclusions than Myo5b KO mice, suggesting a critical role for Pacsin 2 in inclusion formation. n = 3 mice per group. Scale bars = 50 µm. One-way ANOVA was performed with the Bonferroni post hoc test for C; error bars are SEM. *, P < 0.05 compared to Myo5b KO mice.