Trans blot sd semi dry transfer cell

After SDS-PAGE, the proteins in the gel were transferred onto a membrane (Immobilon P, IPVH00010, Merck Millipore) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked with 5% skim milk in PBS, supplemented with 0.05% Tween 20 (PBS-T). The membranes were incubated with the primary antibody: mouse anti-GAS7 (clone 2F6, TA501756, OriGene), anti-Grb2 (Sc-8034, OriGene), anti-Nck (SC-20026, Santa Cruz Biotechnology), anti–N-WASP (#4848, Cell Signaling Technology), anti-WASP (#48606, Cell Signaling Technology), anti-SPIN90 (Ab88467, Abcam), anti-mCherry (71615, Cell Signaling Technology), and anti–glyceraldehyde-3-phosphate dehydrogenase (SC16657, Santa Cruz Biotechnology) at a 1:10,000 dilution, followed by an anti-mouse or anti-rabbit IgG alkaline phosphatase conjugate (Promega) secondary antibody in PBS-T at a 1:10,000 dilution. The alkaline phosphatase was detected by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Roche).

After electrophoretic separation of the proteins, the gels were electrotransferred to polyvinylidene fluoride membranes (Millipore) using a Trans-Blot SD Semi-Dry transfer Cell (Bio-Rad) at 15 V for 35 min. Prior to transfer, the membranes were processed as indicated by the manufacturer. After transferring the proteins, the membranes were blocked for 1 h at room temperature in the presence of 2% bovine serum albumin in PBS mixed with 0.15% Tween20. Polyclonal antibodies against SEPT4_Tsm (1:5,000) were reacted against total protein extracts of the T. crassiceps ORF strain and of T. solium cysticerci. Commercial secondary goat anti-rabbit IgG (H + L) antibody conjugated to horseradish peroxidase (HRP) (1:2,000, 81-6520; Zymed, South San Francisco, CA, USA) and goat anti-mouse IgG (H + L) antibody conjugated to HRP (1:1,000, 81-6520; Zymed) were used to detect binding. Antigen-antibody interactions were revealed by adding a chemiluminescence developmental kit solution (SuperSignal West Pico Chemiluminescent Substrate, Thermo-Fisher Scientific). Membranes were imaged using a ChemiDoc XRS system (Bio-Rad).

To make sure the ar1b inserted into the HearNPV genome was active, both reverse transcription PCR (RT-PCR) and western blot analysis were used to detect expression of ar1b. The total RNA was extracted from Ar1b-HearNPV infected HzAM1 cells (2 × 10⁶) with RNAiso plus (TAKARA) according to the manufacturer’s protocol. Briefly, the cDNA was synthesized using 1.0 μg of total RNA (as template) with the reverse transcriptase M-MLV (Thermo Fisher Scientific, USA) and an oligo(dT) anchor primer. Primers qβ-actin-F and qβ-actin-R (Table 1) were used to detect the β-actin transcription; this was selected as a reference gene. The ar1b transcription level was monitored by primer pairs qAr1b-F and qAr1b-R (Table 1).
Ar1b-HearNPV-infected HzAM1 cells were used to extract the total protein according to the manufacturer’s guidelines [29 ]. Protein samples were then separated by SDS-PAGE (15%) and electro-transferred by Trans-Blot SD semi-dry transfer cell (BIO-RAD) to the nitrocellulose membrane. Immunoblotting was performed by using standard protocols. The primary antibodies used included a monoclonal anti-actin antibody (1:10000) and a monoclonal anti-HA (1:4000), following the secondary antibodies HRP-conjugated goat anti-mouse lgG (1:5000). The proteins were visualized with an enhanced chemi-luminescence system (ECL; GE).

Lysate aliquots containing 50 μg of proteins were denatured, separated on a 4–10% SDS-polyacrylamide gel, and transferred to nitrocellulose membranes using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). Membranes were blocked in 5% non-fat milk and incubated overnight at 4 °C with primary antibodies: anti-GPR151 (LSBio # LS-B6760-50, 1:500 dilution) or anti-beta-actin (Cell Signaling #3700, 1:1000 dilution). Subsequently, the membranes were incubated for 1 h at room temperature with IRDye® 800CW goat-anti-mouse antibody (LI-COR #926-32210, 1:2500 dilution). Target proteins were visualized using Odyssey® Fc Imaging System (LI-COR). Uncropped blots were provided in the Source Data.