Isopropyl β d 1 thiogalactopyranoside (iptg)

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.

For protein expression, plasmids were transformed into Escherichia coli BL21 (TransGen Biotech, CD901-02) cells grown in LB media with 1000 μg/mL ampicillin. After 16 h of expression under 0.5 mM IPTG (BioFroxx, 1122GR005) at 20 °C, the cell pellets were collected, resuspended, and lysed in lysis buffer (5 μg/mL PMSF, 10 μg/mL Triton X-100). This denatured suspension was clarified by centrifugation at 12,000 × g for 10 min at 4 °C and sonicated.
Supernatants containing fusion proteins were loaded onto a His GraviTrap column (ThermoFisher, 88,221) or Glutathione Agarose (ThermoFisher, 1600). The eluted proteins were then concentrated, and the protein concentration was measured via a BCA assay and absorbance.
All liquid droplet formation assays were performed in 150 mM KCl (unless specified), 5 mM MgCl2, 10 mM cAMP (as indicated), 20 mM HEPES, pH 7.0, 1 mM EGTA, 1 mM DTT, 0.5 mM ATP, and 100 mg/ml polyethylene glycol 8000 (unless specified). Purified proteins were incubated at different stoichiometries and at various concentrations at room temperature for 1 h and imaged under DIC and/or fluorescence microscopy.

LAP is composed of 249 amino acids (Human TGF-β1 proprotein, P01137, 30-278). The truncated LAP fragments are No. 1 (1-132), No. 2 (1-65), No. 3 (1-100), No. 4 (45-132), No. 5 (77-249), No. 6 (77-193) and No.7 (194-249). Primers were designed and added with EcoRI and XhoI restriction sites. The target fragments were amplified by PCR and digested. The digested products were inserted into the PET-28a vector to construct recombinant plasmids with a His-tag at the N-terminus. The recombinant plasmids were transformed into E. coli C43(DE3) competent cells. Positive clones were cultured overnight at 37 °C in 3 mL LB medium. The bacterial liquid was inoculated into 20 mL of LB medium and cultured to an OD600 of 0.8. Then the bacterial liquid was added with 0.2 mM IPTG (Biofroxx, Germany) and incubated at 16 °C for 16 h. The collected cells were resuspended with 500 μL Buffer A (25 mmol Tris 7.5, 300 mmol NaCl, 2 mM PMSF) and sonicated on ice. The supernatant was collected by centrifugation and combined with 30 μL of Ni–NTA agarose at 4 °C for 1 h. 500 μL Buffer B (25 mmol Tris 7.5, 300 mmol NaCl, 15 mmol Imidazole) was added to wash Ni–NTA agarose for 3 times. Protein expression was detected by 12% SDS-PAGE.