(A) Probes used to measure MLCK210-dependent barrier loss. (B) Intestinal permeability to fluorescein was evaluated on d14 after syngeneic (B6→WT) or allogeneic (129→WT or MLCK^–/–) BMT. Values are normalized to the mean of WT mice that did not receive BMT. Each point represents an individual mouse. *P < 0.05, 2-tailed t test. (C and D) Intestinal permeability to 4-kDa dextran on d14 (C) or d35 (D) after BMT, normalized to the mean of WT mice without BMT. Each point represents an individual mouse. *P < 0.05, 2-tailed t test. (E and F) Jejunum harvested on d35 and immunostained for phosphorylated myosin light chain (pMLC, green) and E-cadherin (ECAD, red). Arrowheads denote perijunctional actomyosin ring. Note the preservation of myosin light chain phosphorylation within villus smooth muscle in MLCK^–/– mice. Each point represents the average of 4 fields from one segment of tissue; 2 segments were analyzed per mouse. Data are normalized to the mean of WT mice without BMT. Scale bars: 50 μm (top), 10 μm (bottom). **P < 0.01, ANOVA with Bonferroni’s correction (vs. all other conditions). (G and H) 35 days after BMT, mice were injected with Alexa Fluor 647–BSA and sacrificed 30 minutes later. Confocal imaging demonstrates bright intravascular signal, indicative of retained dye, in WT and MLCK^–/– mice without BMT or after syngeneic (B6→B6) BMT. In contrast, intravascular signal is markedly diminished in both WT and MLCK^–/– mice after allogeneic (129→B6) BMT. Note the sharp paracellular pattern in MLCK^–/– mice, consistent with preservation of the tight junction barrier, relative to the diffuse signal over the epithelium of WT mice (arrowheads). Scale bar: 25 μm. Each point represents an average of 6 regions of lamina propria from one mouse. Data are normalized to mean of WT mice without BMT. **P < 0.01, ANOVA with Bonferroni’s correction (vs. other conditions for each genotype; matched conditions across genotypes were not significantly different).