Anti v5 tag

Protein lysates were extracted using RIPA lysis buffer (Pierce, Thermo Fisher Scientific). Protein estimation was conducted using a BCA protein assay kit (Pierce). Protein lysates were denatured and subjected to SDS-PAGE electrophoresis. Proteins were then transferred onto a nitrocellulose membrane and blocked with 5% non-fat milk, followed by the incubation with primary antibody at 4 °C overnight. Membranes were then rinsed and incubated with a corresponding secondary antibody (1:5000, Invitrogen) at room temperature for 1 h. Signal was visualized using Pierce ECL plus Western blotting substrate (Pierce). The following primary antibodies were used in this study: anti-POU1F1 (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HA tag (1:2000; Invitrogen); anti-V5 tag (1:2000; Invitrogen); anti-CD163 (1:5000; Abcam); anti-CD206 (1:5000; Sigma–Aldrich, St Louis, MO, USA), anti-CD11b (Novus Biologicals, Centennial, CO, USA); anti-VEGFA (1:5000; Santa Cruz); anti-p-CXCR4 (1:1000; Abcam, Cambridge, UK); anti-CXCR4 (1:1000; Abcam); anti-p-Akt (1:1000; Cell signaling technology, Beverly, MA, USA); anti-Akt (1:1000; Cell signaling technology); anti-p-VEGFR2 (1:1000; Cell signaling technology); anti-VEGFR2 (1:1000; Cell signaling technology); anti-GAPDH (1:3000; Santa Cruz).

S2 cells were transfected using Cactin-V5 expressing plasmids and then infected with DCV (MOI=1) for 48 h, if necessary. For IP, cells (5×10⁷) were collected and suspended in lysis buffer (2 mM EDTA, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 20% glycerol) supplemented withPhenylmethanesulfonyl fluoride (PMSF) (Sangon Biotech) on ice for 30 min. After centrifugation at 13,000 rpm for 10 min at 4°C, the lysates were incubated for 4 h with the anti-V5 tag antibody (Invitrogen) at 4°C, followed by further incubation with Protein A/G Agarose for1h. The beads were washed five times with lysis buffer. The proteins were eluted by boiling the beads for 5 min in 1 × SDS loading buffer and analyzed by Western blot with the anti-V5 tag (Invitrogen) or anti-p-Ser/phosphoserine (Santa Cruz).