Syt2 and Syt1 are differentially expressed at the mouse diaphragm and bladder.

A. Immunohistochemistry analysis detected expression of Syt2, but not Syt1, in motor nerve terminals at WT mouse diaphragm neuromuscular junctions (NMJs). SMI-312 antibody detects neurofilament and marks the phrenic nerve. α-Bungarotoxin (a-btx) labels post-synaptic acetylcholine receptors and serves as a marker for NMJs. Scale bar, 20 μm. B. Immunohistochemistry analysis of bladder sections from WT mice detected expression of Syt1, but not Syt2 in neurons. β-3 tubulin marks nerve fibers and synapsin marks the pre-synaptic site of neuronal varicosities in bladder tissues. Scale bar, 20 μm. C. Diaphragms isolated from WT, Syt1^M3 KI, or Syt2^M3 KI mice were incubated with BoNT/B (100 nM, 90 min) in high K⁺ buffer. Tissues were washed, and immunohistochemistry analysis was performed on whole mount tissues. BoNT/B binding and entry was detected on both WT and Syt1^M3, but not Syt2^M3 diaphragms. D. Bladder tissues isolated from WT, Syt1^M3, or Syt2^M3 mice were incubated with BoNT/B (100 nM, 90 min) in high K⁺ buffer. Tissues were washed, and immunohistochemistry analysis was performed. BoNT/B binding and entry was detected on both WT and Syt2^M3, but not Syt1^M3 bladders. Representative images were selected from n = 3–4 biological replicates.