SDS-PAGE and Western Blot analysis
Larval extracts and purification fractions were resolved by SDS-PAGE
(10% polyacrylamide gels). Before loading the samples into the wells,
they were heated for 5 min at 100 °C in sample buffer (125 mM Tris–HCl,
pH 6.8, 4% [w/v] SDS, 20% [w/v] glycerol, 0.01% [w/v]
bromophenol blue, and 10% [v/v] 2-mercaptoethanol). One of the
lanes was reserved for the protein marker to determine the MW of the
protein bands. The resulting gels were either stained with Coomassie
Blue R-250 or transferred onto nitrocellulose membranes (Cytiva,
Marlborough, USA). Membranes were then incubated overnight at 4 °C in
blocking solution (phosphate-buffered saline [PBS]—3% skim milk
[PBS-M]). After a 10-min wash with PBS containing 0.05% v/v Tween
20 (PBS-T), the membrane was incubated for 2 h with anti-his antibody
(BD Biosciences, USA) 1/2,500 in 0.05% PBS-T-1% skim milk, and then
washed three times with PBS-T. Polyclonal rabbit anti-mouse
immunoglobulin conjugated with HRP (1/30,000 in 0.05% PBS-T-1% skim
milk) was used as the secondary antibody. Development was carried out
with 3,3’-diaminobenzidine (DAB) (Sigma-Aldrich, USA) staining or,
alternatively, with an enhanced chemiluminescent substrate (ECL) and
high-performance chemiluminescence films (CL-X Posure™, Thermo Fisher
Scientific, USA).