Dynapro

Purified PI3KIα enzyme was purchased from Invitrogen-Life Technologies. Purified PI4KIIα was purchased from Creative Biomart. Dynamic light scattering data were recorded on a Wyatt DynaPro dynamic light scattering plate reader. Endogenous PtdIns(4,5)P₂ (brain, porcine) and phosphatidylinositol (PI) 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) were purchased from Avanti Polar Lipids. ATP and TLC plates with silica gel 60 were purchased from Sigma. All solvents were purchased from Fischer Scientific.

The hydrodynamic radii (R_h) of the ELP₁₆₀–srt–ABD–Dox and KEKE–ELP₁₆₀–srt–ABD–Dox conjugates were determined by DLS (DynaPro; Wyatt Technology, Santa Barbara, CA) at 25 °C using a single detector at 90°. Samples were prepared in sortase reaction buffer at 25 × 10⁻⁶ M, filtered through 0.22 μm Millex-GV filters (Sigma-Aldrich, St. Louis, MO), and measured using a DynaPro plate reader (Wyatt Technology, Santa Barbara, CA). The data were analyzed with a regularization fit of the autocorrelation function using a Rayleigh sphere model. The purity of the drug conjugates (KEKE–ELP₁₆₀–ABD–Dox and ABD–Dox) and efficiency of the sortase enzymatic reaction were assessed by size exclusion HPLC. Samples were injected into a LC10 HPLC (Shimadzu Scientific Instruments, Columbia, MD) with a Shodex OHPak SB-804 column (New York, NY) and PBS:acetonitrile (70:30 v/v) as the mobile phase at an isocratic flow rate of 0.3 mL min⁻¹. Eluting peaks were detected with a UV–vis detector set at 488 nm. Mass spectrometry analysis of the ABD–Dox conjugate was performed on a Bruker Autoflex Speed MALDI-TOFMS (Bruker Daltonics, Billerica, MA) using succinic acid matrix and porcine insulin as the internal standard.