Ripa lysis and extraction buffer

Mice were perfused with PBS prior to enucleation of eyes. Eyes were processed to remove the anterior segment, lens, and neural retina. The remaining posterior eyecups were flash frozen with liquid nitrogen before processing. Frozen eyecups were homogenized using 80 ul of RIPA lysis and extraction buffer (#PI89901, VWR, Radnor, PA) with ethylenediaminetetraacetic acid (EDTA)-free protease inhibitors (#11836170001, Sigma-Aldrich, St. Louis, MO). Cholesterol was measured using a colorimetric Total Cholesterol Assay Kit (#STA-384, Cell Biolabs, San Diego, CA). Cholesterol values were normalized to the weight of the individual eyecup.

Primary mouse gingival fibroblasts were cultured in DMEM supplemented with 10% FBS and 100 U penicillin/0.1 mg/ml streptomycin in the presence of 0, 0.1, 1 or 10 µg/ml of CpG ODN at 37 °C, 5% CO₂ in a humidified incubator for 24 hours.
Culture supernatant or cell solution lysed with RIPA Lysis and Extraction buffer (VWR, Radnor, PA, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The Ctsk protein concentration in the culture supernatant or lysed cell solution was determined by a colorimetric method (OD at 450 nm) using a Ctsk ELISA kit (MyBioSource, San Diego, CA, USA). Statistical analysis was performed using one-way analysis of variance with Bonferroni’s multiple-comparison test to assess the difference only of pairs relative to the control group. P < 0.05 was considered as statistically significant.