Picolorlock phosphate detection reagent

Release of phosphate from potato amylopectin was determined as done previously (23 (link), 24 (link), 42 (link), 93 (link)) with the following modifications. Briefly, phosphate release was monitored using the PiColorLock Phosphate Detection Reagent (Novus Biologicals), a malachite green–based assay. For time course assays, 5 nM recombinant protein was incubated with 90 μg solubilized potato amylopectin (Sigma-Aldrich), supplied as a powder; solubilized at a stock concentration of 5 mg/ml using alcohol/alkaline method (also referred to as the “Roach method” in Ref. (93 (link))) in phosphatase buffer in a final volume of 80 μl at pH 6.5. For TgLaforin kinetic characterization, 5 nM TgLaforin was incubated with varying amylopectin concentrations for 10 min. All reactions were terminated by addition of 20 μl (0.25 initial reaction volume) of the PiColorLock Gold solution containing Accelerator in a 100:1 ratio of Gold solution to the accelerator. After 5 min, 8 μl stabilizer solution (0.1 initial reaction volume) was added, and reaction was allowed to develop for 30 min at r.t. before the absorbance of each reaction was measured at 635 nm using a Synergy HTX Multi-Mode Reader (BioTek). Absorbance was converted to pmol P_i release using a P_i absorbance standard curve. Data points are presented as the mean of three independent replicates, each consisting of three technical replicates.

Purified flagellin from P. aeruginosa (PA FLA) encoded by the fliC gene [19 (link)], and sweet potato PAP were purchased from InvivoGen (Sandiego, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Both were reconstituted in endotoxin-free water (Sigma-Aldrich, USA). PiColor Lock phosphate detection reagent was purchased from Novusbio (Centennial, CO, USA) while the Cymax Human IL-8 ELISA kit was obtained from Ab Frontier (Seoul, Korea). The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (Total) InstantOne ELISA kit was procured from Invitrogen, Thermo-Fisher Scientific (Carlsbad, CA, USA).
The human alveolar carcinoma epithelial cell line (A549 cells) was procured from American Type Culture Collection (ATCC CCL-185) and was maintained at F-12K Medium Ham’s F-12K (Kaighn’s) Medium (Gibco Life technologies; Gaithersburg, MD, USA) with 10% fetal bovine serum (Gibco Life technologies, USA) and 1% penicillin-streptomycin (Gibco Life technologies, USA) at 37°C in a 5% CO₂ humidified incubator.
HEK-Blue hTLR5 cells (Invitrogen, Thermo-Fisher Scien tific, USA) were cultured at 37°C in Dulbecco modified eagle medium (DMEM; Gibco Life technologies, USA) with 30 μg/mL of blasticidin (Invitrogen, Thermo-Fisher Scientific, USA) and 100 μg/mL of Zeocin (Invitrogen, Thermo-Fisher Scientific, USA).