Anti fyn antibody

To investigate the interaction between 2B4 and SHP-2 or Fyn in the infected group with or without anti-2B4 antibody treatment, we used an anti-2B4 (Cell Signaling Technology, USA), anti-SHP-2 (Abcam, England), or anti-Fyn antibody (Abcam, England) for immunoprecipitation (IP). Cells from the uninfected group, infected group, and anti-2B4 antibody-treated infected group were collected and lysed in IP lysis buffer (Beyotime Technology, China) supplemented with protease inhibitors (Beyotime Technology, China) on ice for 45 min and centrifuged at 12,000 rpm at 4 °C for 15 min. Next, the supernatants were collected and incubated with 1 µg of the appropriate antibody at 4 °C overnight and precipitated with protein A/G-agarose beads (Beyotime Technology, China) for another 7 h at 4 °C. The beads were then washed with lysis buffer three times by centrifugation at 12,000 rpm at 4 °C. The precipitated proteins were denatured in 5 × loading buffer and analyzed by Western blotting.

Expi293F cells transfected with p-SELECT-zeo-rISRAA were harvested after 48 hours and lysed. Pull-down assays were performed using the Pierce His Protein Interaction Pull-Down Kit (Thermo Fisher Scientific, MA, USA) according to manufacturer’s recommendations. Briefly, equilibrated cobalt resin beads were incubated with Expi293F cell lysates for 30 min on ice. Untransfected cells were used as a negative control. The beads were washed 5 times and stored. A bead fraction was boiled in SDS buffer and then analyzed for rISRAA immobilization via western blotting using a polyclonal rabbit anti-His-Tag antibody at a final dilution of 1:1000 (Cell Signaling Technology, Danvers, MA, USA) and an anti-rISRAA monoclonal antibody used at a final dilution of 1:100 (GenScript, Piscataway Township, NJ, USA). EL4 cell lysates were prepared and incubated with rISRAA immobilized beads for 2 hours with gentle rotation at 4°C. After washing, the protein complexes were eluted from the beads with 300 mM imidazole. The eluted fractions were analyzed for Fyn protein detection via western blotting using a rabbit polyclonal anti-Fyn antibody (Abcam, Cambridge, UK) used at a final concentration of 1:1000.