Versadoc imager

Total protein was extracted from 6-mm RPE/choroid punches obtained from four donors homozygous for the CFH Y allele, five donors homozygous for the H allele, and five heterozygous donors, and concentrations were determined using the BioRad DC Assay according to the manufacturer’s instructions (BioRad, Hercules, CA, USA). For each sample, 20 μg of protein was diluted in PBS with 2 mm CaCl2 and 140 mm NaCl mixed with Native Sample Buffer (BioRad) and loaded into a 10% Mini-PROTEAN TGX precast gel (BioRad). Semi-native PAGE separation was performed using 0.005% SDS. Proteins were then blotted onto a polyvinylidene difluoride (PVDF) membrane (BioRad). Membranes were treated briefly with methanol followed by blocking with 1% BSA (Research Products International, Mt Prospect, IL, USA) in PBS with 0.1% Tween-20 (PBS-T; Research Products International). To detect both forms of CRP, the membrane was incubated with primary antibodies directed against pCRP (1D6) or mCRP (3H12) for 2.5 h. Following three 5-min washes in PBS-T, membranes were treated with Alexa Fluor® 488-conjugated donkey anti-mouse secondary antibody (A-21202; Thermo Fisher Scientific, Waltham, MA, USA) and washed three times for 5 min each in TBS with 0.1% Tween-20 (TBS-T; BioRad). Finally, bands were detected using a VersaDoc Imager (BioRad).

Total RNA was extracted from control and treated cells using TRIzol reagent (Life Technologies, Inc., Carlsbad, CA) according to manufacturer instructions and the cDNA synthesis was carried out as described by us earlier (29 (link)). About 2 µg of template cDNA was used for cycling in a thermal cycler (Thermo Fisher, Pittsburgh, PA). The PCR products were separated on a 1.5% agarose gel, stained with 0.5 mg/mL ethidium bromide, and visualized under UV light using BioRad Versa Doc imager.