Protein assay

Hexahistidine tagged, signal peptide deficient versions of PLY, PFO and SLO and associated derivatives were expressed in E. coli Tuner cells. Cells were grown, and the recombinant proteins purified and stored as described previously [71] (link). Fluorescently labeled proteins were stored as described [71] (link), in the absence of reducing agent. Prior to use all proteins were centrifuged at 14,000×g (Eppendorf, Centrifuge 5417R) to remove any precipitated proteins and assayed for protein concentration using the Bio-Rad protein assay with bovine serum albumin (BSA; Sigma) as a standard.

RAW 264.7 and ARPE-19 cells and ocular tissues were lysed in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.4), 0.1 M ethylenediaminetetra acetic acid (EDTA), 10 mM NaCl, and 0.5% Triton X-100] supplemented with a protease and a phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined by protein assay (ThermoFisher Scientific) using BSA (Sigma-Aldrich) as a standard. Lysates (20 μg) were resolved on a 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to PVDF membranes (BioRad, Hercules, CA, USA). After blocking with Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.1% (w/v) Tween 20, membranes were probed with primary antibody. The following specific antibodies were used to characterize protein expression: iNOS, COX-2, (p-)AKT, (p-)ERK1/2, (p-)JNK, (p-)p38, (cleaved-)Caspase 3/8, (cleaved-)PARP1, and β-actin. After overnight incubation at 4 °C, then incubated with secondary antibody (anti-rabbit IgG-HRP or anti-mouse IgG-HRP). Immunoblots were visualized using chemiluminescent (ECL) Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) and immunoreactive signals were analyzed by densitometry scanning (Amersham imager 600, GE Healthcare, Buckinghamshire, UK).