Anti mbp

At each time point, 30 larvae from control or metronidazole treatment groups were harvested in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, 78830), Protease Inhibitor Cocktail (Promega, G6521), Phosphatase Inhibitor Cocktail 2 (Sigma, P5726), and Phosphatase Inhibitor Cocktail 3 (Sigma, P0044). Western blotting was performed using standard protocols [48 (link)]. Eleven primary antibodies were used in this study: anti-IL-7R (1:1500; Abcam, ab180521), anti-MBP (1:500; Anaspec, #55811, Fermont, CA, USA), anti-Phospho-JAK1 (1:1,000; Cell Signaling Technology, #3331, Danvers, MA, USA), anti-Phospho-JAK3 (1:500; Cell Signaling Technology, #5031), anti-STAT3 (1:500; Santa Cruz Biotechnology, sc-7179, TX, USA), anti-Phospho-STAT3 (1:200; Cell Signaling Technology, #9145), anti-STAT5 (1:500; Santa Cruz Biotechnology, sc-836), anti-Phospho-STAT5 (1:200; Cell Signaling Technology, #9351), anti-BCL2 (1:1000; BD Biosciences, 610538), anti-CASPASE-3/cleaved CASPASE-3 (1:300; Wanleibio, WL02117), and anti-CASPASE-7/cleaved CASPASE-7 (1:500; Wanleibio, WL0181). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:3,000; Millipore, MAB374, Billerica, MA, USA) was used as a loading control.

Western blot analysis was performed to test the expression of MBP protein. Ten larvae were lysed in buffer with protease inhibitors (Complete Mini, Roche, Mannheim, Germany), and the protein concentrations were quantified with a BCA Protein Assay Kit (Complete Mini, Roche, Mannheim, Germany). The proteins were separated in a 10% SDS-PAGE gel and were transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then blocked in 5% nonfat dry milk in PBS for 1 hour and incubated with anti-MBP (1∶500; Anaspec, Fermont, CA) and anti-GAPDH (1∶3,000; Millipore). The blots were rinsed with PBS and incubated with peroxidase-conjugated goat anti-rabbit antibodies (1∶5,000; Sigma-Aldrich, St. Louis, MO) for 1 hour. The bound antibody was visualized using an enhanced chemiluminescence assay (Millipore).