Bl21 de3 competent cells

All gene fragments were from Synthetic (Engineering, Xian, China) and subcloned in the protein expression vector PET-32a (Life Technologies). Recombinant plasmids were analyzed using DNA sequencing for double strands to confirm that no mutation was introduced in both clones. Correct plasmids were then transformed into BL21 (DE3) competent cells (Vazyme, Nanjing, China) for protein expression. When the A600 of the Escherichia coli culture reached 0.6–0.8, the proteins of interest (i.e. EGFP, EGFP + RAAS, RAAS + EGFP, CD2v 231-300 aa+ 6xHis, and CD2v 231-319 aa+ 6xHis) were expressed by adding 0.1 mM isopropyl β-d-Thiogalactoside to the culture for 12 h at 16 °C. The recombinant fusion protein was purified using Ni affinity chromatography. The protein concentration was quantified using the Nanodrop one 2000 (Thermo Fisher Scientific, Massachusetts, United States), after which the purified protein (50 μM) was incubated with CHO in 35 mm glass-bottomed Petri dishes (NEST, Wuxi, China). Finally, cells were washed thrice with PBS to remove unbound proteins and imaged using confocal microscopy.

The pGEX-4T1 constructs were subjected to BL21(DE3) competent cells (Vazyme) for transformation and then prokaryotic protein expression was induced with 0.2 mM IPTG (BioFroxx) at 37 °C for 3 h. After the bacterial precipitates were lysed and ultrasonically crushed, prokaryotic HOXA10 mutant proteins were purified by mixing the precipitates with Pierce Glutathione Agarose (Thermo). Finally, the beads were eluted with GSH (Sigma) overnight to obtain the purified protein.