Amicon ultra 15 10000 mwco centrifugal filters

In some experiments, the intracellular levels of Profilin were modified using a membrane permeable version of PfnI [24] (link), [40] . PTD4-Profilin (21 kDa) was generated by fusing a transduction domain, PTD4 [41] (link), to Profilin. The recombinant protein was expressed in E. Coli and purified as described previously [40] . Briefly, competent E. Coli BL21-PLys transformed with the pRSETA-PTD4-Pfn I vector were induced by adding 1 mM IPTG (Sigma) at 37°C for 6 h. Bacterial pellets were lysated by freezing and thawing protocol in liquid N₂, followed by sonication on ice in the presence of DNAse and a protein inhibitor cocktail (Sigma). Cellular lysates were resolved by centrifugation, and the soluble protein was isolated by employing Ni-NTA resin-packed columns (Quiagen). Protein wash and elution was carried out with high concentrations of imidazole. Buffer exchange and concentration of the recombinant protein were performed by centrifugation in Amicon Ultra-15 10000 MWCO centrifugal filters (Millipore), replacing the elution media with PBS. Proteins were frozen in liquid N₂ and stored at −80°C in 10–15% glycerol-PBS. Bacteria and proteins were handled according to the Safety Guidelines for Laboratory Personnel Working with Trans-Activating Transduction (TAT) Protein Transduction Domains.

The total RNA of PSCs was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse transcribed into cDNA using the ThermoScript™ RT-PCR System for First-strand cDNA Synthesis Kit (Invitrogen). The nucleotide sequence encoding the LEL domain of Eg-TSP1 was amplified from cDNA using a sense primer (5′-CGCGGATCCCCTGATAACCTAAACAAAGC-3′) containing a BamHI site (underlined) and an antisense primer (5′-CCCAAGCTTGAGGGTTTTGTTCTCTGCCAA-3′) containing a HindIII site (underlined). The PCR products were ligated into the pET32a(+) plasmid (Novagen, Madison, WI, USA). The recombinant plasmid with correct sequence was transformed into Escherichia coli BL21 (DE3) cells (Invitrogen) and subsequently grown at 37 °C in Luria-Bertani broth containing 50 μg.mL⁻¹ ampicillin. When the OD_600nm value of bacteria reached 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for 5 h induction at 37 °C. The recombinant protein was harvested from E. coli lysates, followed by purification with a Ni²⁺ affinity column (Bio-Rad, USA) under denaturing conditions in 8 M urea. The purified protein was refolded by dialysis against PBS and concentrated using an Amicon Ultra-15 10,000 MWCO centrifugal filters (Millipore, USA), and then analyzed by SDS-PAGE.