Electrochemiluminescence

Following the method detailed in a previous study (Castro-Sesquen et al., 2014 (link)), the 10 μg purified LPG or T. brucei total lysate was mixed with 1 × sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) loading buffer and then heated at 100°C for 5 min. The samples were separated on 10% Tris-Glycine polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, United States) using a Mini Trans-blot system (Bio-Rad, Hercules, CA, United States). After being blocked with 5% (w/v) skim milk in PBS for 1 h at 37°C, the membranes were incubated overnight with mouse monoclonal anti-LPG IgM (1:1,000; Cedarlane) and healthy mouse IgM as a control. After washing with PBS-Tween20 (PBST), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgM (1:1,000; Sangon Biotech, Shanghai, China) for 1 h at 37°C. Finally, the samples were visualized using electro-chemiluminescence (Solarbio).

RIPA lysis buffer (Solarbio, Beijing, China) to extract total protein. The proteins were quantified by the bicinchoninic acid (BCA) assay kit (Solarbio, Beijing, China). Add SDS loading buffer to the extracted total protein, and then boil it in 100° water for five minutes for subsequent experiments. The obtained protein was electrophoresed in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). PVDF membrane was used for electroporation. The protein electrophoresis was performed at a stable voltage of 100 V, and the electroporation was performed at a stable current of 300 mA for 90 min. After electroporation, soak the PVDF membrane in 5% skimmed milk and place it on a shaker for half an hour. Then add 5 ml of CCND2 (1:1000), IGF2BP3 (1:1000) (Abcam, CA, USA), RB (1:500), and GAPDH (1:5000) (Cell Signaling Technology, MA, USA) rabbit-derived primary antibody to the PVDF membrane and incubate overnight at 4°. After incubating overnight, collect the primary antibody, add Tris-Buffered Sal ine Tween 20 (TBST) and wash three times for 15 min each time. Add goat anti-rabbit (Solarbio, Beijing, China, 1:5000) and incubate for 1 h, then continue to wash with TBST three times. Finally, add electrochemiluminescence (Solarbio, Beijing, China) liquid to expose in the exposure instrument. The antibodies used in the experiments are shown in Additional file 2: Table S2.