Protease and phosphatases inhibitor cocktail

Cell replating assays were performed on CD cells that were trypsinized, washed, suspended in serum-free DMEM, plated on LM-332 or LM-511 (1 μg/ml) and harvested at 0, 30 and 60 min later. Cells were washed in PBS and lysed using M-PER reagent with protease and phosphatases inhibitor cocktails (Sigma). Protein extracts (20–40 μg) were subjected to Western immunoblot analysis.

CD cells grown on LM-511 for 1 h were stimulated with FGF10 or GDNF (10 ng/ml each) and harvested at 0, 5, 15 and 30 min later. Cells were washed in PBS and lysed using M-PER reagent with protease and phosphatases inhibitor cocktails (Sigma). Protein extracts (20-40 μg) were subjected to Western immunoblot analysis. When chemical inhibitors were used they were added 1 h prior to the assays. For silencing experiments, cells were transfected with non-silencing siRNA (20 nM, transfection control) or TRAF6 siRNA (20 nM) using Lipofectamine RNAiMAX according to manufacturer’s instructions. Transfected cells were utilized 24 h later. For experiments using adenoviruses, cells were infected with ad-GFP, ad-Δp85 or ad-myrAkt for 48 h prior to treatments.

Healthy (control) and LSCD-diseased rabbit corneal epithelial cells were harvested by exposing the corneas to 20% isopropyl alcohol. The corneas were then washed three times with saline, and epithelial cells were scraped and removed from the corneal surface, using a small scalpel blade. Healthy rabbit corneal epithelial cells (H-CEC) were collected the day of the lamellar limbectomy that induced LSCD. LSCD-diseased epithelial cells (D-CEC) were collected approximately 3 months after limbectomy on the day of CAOMECS grafting. Both epithelial cell samples (from the same rabbits) were then used for proteasome biochemical analysis. The cells were suspended in lysis buffer containing 20 mM Tris-HCl pH = 7.5; glycerol 10% EGTA 1 mM; DTT 1 mM; protease and phosphatases inhibitor cocktail as instructed by the supplier (Sigma, St Louis, MO). Cell membranes were disrupted by pipetting 25 times the cells trough a hypodermic 18G needled.