Anti β actin antibody

SDS-PAGE and Western blotting were carried out as previously described by Liu et al.’s protocol (2017). Each protein sample was mixed with sample buffer containing SDS, DTT and 5% β-mercaptoethanol (β-ME) and then boiled at 100°C for 10 min; then each sample was separated by 10% SDS-PAGE (Toolsbiotech, Taiwan) under reducing conditions. Next the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, USA) using a transfer apparatus. Anti-β-actin antibody (Santa Cruz Biotechnology Inc., USA), secondary anti-mouse immunoglobulin-horseradish peroxidase (PerkinElmer, Inc., USA), anti-histone H2B antibody (Cell Signaling Technology, USA) and secondary anti-rabbit immunoglobulin-horseradish peroxidase (KPL, USA) were used for the Western blot analysis. For the production of anti-Ascec B2 antibody, the most variable regions of the peptide sequence across the five Ascecs and Ae. aegypti cecropin B (Aacec B) were identified by BLAST and antigen prediction. Specifically, the peptide sequence region near the C-terminal end (amino acid sequence N-VNAAQKGLPVAAGIQALGR-C) was identified and used to generate an appropriate antigenic peptide, which was then used for rabbit polyclonal anti-Ascec B2 antibody generation by Yao-Hong Biotechnology Inc. (Taiwan).

Cells were isolated from DRG and made to single cell suspension. Washed with PBS for 2 times, then added RIPA lysis buffer and maintained on ice for 30 min accompanied with vortexing 30 s per 10 min. Centrifuged for 15 min at 4 °C with 12 000 rpm and transferred the supernatant to a new tube. The protein concentrations were measured using Nanodrop 2000 (Thermo Fisher Scientific, San Jose, CA, USA). Then added 10× loading buffer to the protein solution and heated on 95 °C for 5 min. Proteins (10 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA). After blocked with 5% dried skimmed milk in Tris-buffered saline (TBS), the membranes were incubated with specific primary antibodies including anti-BDNF, anti-Wnt5a and anti-β-catenin (1 : 100 dilution, and all from Santa Cruz Biotechnology) and anti-β-actin antibody (1 : 2000 dilution, Santa Cruz Biotechnology) at 4 °C overnight. The PVDF membrane was washed five times with TBS with Tween-20 (TBST), followed by incubation with HRP-conjugated anti-mouse IgG secondary antibody (1 : 2000 dilution, Santa Cruz Biotechnology) at room temperature for 1 h. After being washed with TBST five times, the protein signals were detected using PierceTM ECL western blotting substrate (Thermo Fisher Scientific).