Rabbit anti rfp antibody

The DUAL membrane yeast two-hybrid system constructed by HiTech (Shanghai, China) was applied to screen ORFV ORF120 interaction proteins. The experimental procedures were carried out according to the manufacturer’s instructions. To investigate the interaction of the ORFV ORF120 protein and host factor G3BP1, OFTu cells were cotransfected with pIRES-puro3-EGFP120 and pCMV-N-myc-G3BP1 or either empty vector. The cells were harvested 24 h posttransfection and lysed. Protein extracts of the transfected cells were immunoprecipitated with mouse anti-myc antibody (number 60003-2-Ig; Proteintech) or mouse anti-GFP (number 66002-1-Ig; Proteintech). The antigen/antibody complex was incubated with protein A/G agarose beads (Thermo Fisher). Finally, the immunoprecipitates were washed three times with lysis buffer and then subjected to Western blotting. Furthermore, immunoprecipitation of G3BP1 was performed on OFTu cells infected with RFP-tagged ORF120 revertant virus using a rabbit anti-RFP antibody (number ab152123; Abcam), and the pcDNA3.1-RFP-Becline 1 plasmid was used as a control.

Western blots were used to examine shRNA knockdown efficiency. N2A cells were co-transfected with the constructs that express Flag-tagged FlRT3 or Flag-tagged CDKN1C and the corresponding shRNAs. After 24 h incubation, untransfected and transfected cells were harvested and lysed for protein extraction. Then the protein extract was run on 10% SDS PAGE gels. Proteins were then transferred to a PVDF membrane. The membranes were treated with blocking reagent (3% bovine serum albumin) for 1 h before incubated with the rabbit anti-RFP antibody (Abcam) overnight in a cold room. After several washes, the membranes were incubated with the secondary antibody (goat anti-rabbit immunoglobulins/HRP), washed again, and developed with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096). Images were collected using a Gel Doc XR+ Gel Documentation System (Bio-Rad) machine. The same membranes were then washed and incubated with the mouse Flag-m2 antibody (Sigma-Aldrich) overnight. On the second day, the membranes were washed, incubated with the secondary antibody (rabbit anti-mouse immunoglobulins/HRP), washed again, developed and exposed in the Gel Doc XR+ Gel Documentation System (Bio-Rad) machine. Details of the antibodies used are provided in Table S4.

Mice were anesthetized with pentobarbital sodium and transcardially perfused with saline followed by 4% paraformaldehyde. Vibratome sections (30 μm) of the spinal cord and brain or Cyrostat-sections (16 μm) of the L4/L5 DRG were immunostained using standard protocol with goat anti-NR1 antibody (Santa Cruz, 1:100), biotinylated isolectin B4 antibody (vector laboratories, 1:200), rabbit anti-CGRP antibody (Calbiochem, 1:500), goat anti-CGRP antibody (Abcam, 1:500), mouse anti-NF200 antibody (Sigma-Aldrich, 1:200), rabbit anti-PSD-95 antibody (Abcam, 1:500), rabbit anti-synaptophysin antibody (Abcam, 1:500), goat anti-GFP antibody (Rockland, 1:500), rabbit anti-RFP antibody (Abcam, 1:500), mouse anti-Flag (Abbkine, 1:800). The number of immunoreactive neurons per DRG section was counted and numbers were averaged over 10 sections per mouse and 4 mice per treatment group. All images were captured with Olympus Fluoview version 3.1 software in an Olympus confocal microscope. See also the list of antibodies used in Supplementary Table 1.