Clone ac 15

Blood samples for Western blotting were obtained from the tail vein and erythrocytes were lysed. Total protein lysates and transfer were performed as described previously [19 (link)]. The following primary antibodies were used: mouse monoclonal anti-HA, clone 6E2 (Cell Signaling, Danvers, USA), mouse monoclonal anti-beta-actin, and clone AC-15 (Sigma-Aldrich, Steinheim, Germany). Secondary goat anti-mouse horseradish peroxidase (HSP)-conjugated antibody was used (Dako Cytomation, Glostrup, Denmark) (see Additional file 7: Table S2).

Neurons were exposed to 100 nM H_C/B in high-K⁺ buffer containing 87 mM NaCl, 56 mM KCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, 0.5 mM MgCl₂, and 1 mM CaCl₂ for 5 minutes at 37°C. Cells were then washed three times with PBS. Binding of H_C/B was examined with two complementary approaches: (1) For immunostaining, neurons were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Images were collected using a Zeiss 880 laser scanning confocal microscope with a 40× oil objective. At least three representative images were collected per condition. (2) For immunoblot analysis, the final concentration of proteins for neuron binding is 0.1 μM, and the total volume is 200 μl. Neurons in each well were lysed in a lysis buffer (PBS with 1% Triton X-100, 0.05% SDS and protease inhibitor cocktail [Roche], 80 μL per well in 24-well plates). The 20-μl 5× loading buffer was mixed with the supernatant of lysed cells. Samples were heated to 56°C for 5 minutes, lysates were centrifuged for 10 minutes at 4°C, and then 5-μl supernatants were subjected to SDS-PAGE and western blot analysis. Binding of H_C/B was detected using a monoclonal human anti-HA antibody. The antibodies used were purchased from the indicated vendors: mouse monoclonal antibody for actin (Clone AC-15, Sigma), mouse monoclonal anti-HA (16B12, Covance), and rabbit polyclonal antisynapsin (Millipore).