S9684

All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-SNAP25 antibody (1/2000; S9684, Sigma-Aldrich). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (1/2000; A6154, Sigma-Aldrich) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India), and intensities of the total form and cleaved form of SNAP25 were measured with GeneTools (Philomath, OR, USA).

The protocol for SNAP-25 was the same as for PLCβ2 (protocol courtesy of Dr. L. Barlow, University of Colorado School of Medicine, Aurora, CO). Tissues were incubated in rabbit anti-SNAP-25 primary antibody (1:5000; Sigma Aldrich cat # S9684; RRID: AB_261576) overnight at 4⁰ C. The concentrations, times of treatment of the Alexa 546 secondary antibody, and Sytox labeling procedures were the same as above.

All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-Firefly antibody (ab185924, Abcam, 1/5000) or anti-SNAP-25 antibody (S9684, Sigma-Aldrich, 1/2000). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (A6154, Sigma-Aldrich, 1/2000) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India).
For SNAP-25 cleavage assay, intensities of the total form and cleaved form of SNAP-25 were measured with GeneTools (Philomath, OR, USA).