Supersignal pico plus chemiluminescent substrate

Protein lysates were prepared using RIPA lysis buffer (Thermo Fisher Scientific), and protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were separated by SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). After blocking, the blot was incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody. Chemiluminescence signal was visualized using SuperSignal Pico PLUS chemiluminescent Substrate (Thermo Fisher Scientific). The following primary antibodies were used in this study: anti-GM130 (1:1000, #12480); anti-CD54 (1:1000, #4915); anti-Annexin V (1:1000, #8555); anti-CD9 (1:1000, #13174); anti-TIMP2 (1:1000, #5738); anti-TIMP3 (1:1000, #5673); anti-GAPDH (1:1000, #5174); anti-VEGFR2 (1:1000, #9698); anti-ERK (1:1000, #4695); anti-p-ERK (1:1000, #4370); anti-MMP2 (1:1000, #87809); anti-MT1-MMP (1:1000, #13130) antibodies were purchased from Cell Signaling technologies (Beverly, MA, USA).

Samples for western blot analysis were prepared essentially as previously described (Ravenscroft et al., 2020 (link)). Briefly, 10–15 flies were homogenized in 1× Laemmli gel loading buffer (30 µL per fly), supplemented with 5 mM EDTA, 1 mM PMSF, and 1× protease inhibitor cocktail (Sigma). Insoluble material was removed by centrifugation 15 min at 18,000 × g, +4°C. Proteins were separated using 4–20% SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad). Membrane was blocked in 5% non-fat dry milk in 1× TBST, pH 8.0 and developed using rabbit anti-GFP (1:4000, Thermo G10362) and goat anti-rabbit-HRP (1:9000, Jackson ImmumoResearch 111-035-0030) primary and secondary antibodies, respectively. For protein loading control, membranes were stained with Ponceau S prior immunostaining. SuperSignal Pico PLUS Chemiluminescent substrate (Thermo 34577) was used to develop the western blots. Chemiluminescent signal was recorded on GE/Amersham I600 imager and quantified using ImageJ.