Phospho p38 p p38

The remaining renal tissue was snap frozen in liquid nitrogen. Kidney samples were homogenised in RIPA buffer for protein analysis by western blotting. Protein concentration was measured before electrophoresing on 12% (w/v) SDS- polyacrylamide gels. After electrotransfer to Immobilon-P membranes (Millipore, USA), membranes were blocked at room temperature with 10% (w/v) milk powder for 1 hour. The primary antibodies against pERK1/2 (1:1000, Cell Signalling, USA), total ERK1/2 (1:1000, Cell Signalling), calnexin (1:1000, Sigma Aldrich, UK), phospho-cJun(p-cjun) (1:1000, Cell Signalling), phospho-p38(p-p38) (1:1000, Cell Signalling) and PAI-1 (1:1000, Abcam, UK) were incubated overnight at 4°C. After washing, the membranes were incubated with goat IgG-horseradish peroxidise conjugated secondary antibodies (DAKO) at a final dilution of 1:20,000 for 1 hour at room temperature. After washing, antibody binding was visualised with enhanced chemiluminescence (Roche, UK) and autoradiography. The expression levels of proteins were analysed on western blots by using 20μg of total cell lysates with loading errors corrected for using calnexin due to its different molecular weight.

Rat corneal proteins were extracted with cold RIPA buffer. Aliquots having equal protein content were subjected to electrophoresis on 9% Tricine gels and then electrophorectially transferred to PVDF membranes. After 1 h blocking in 5% BSA, the blots were incubated with primary antibodies for: MK2(1:1000, Cell Signaling Technology, Danvers, MA, USA), Phospho-MK2(p-MK2, 1:1000, Cell Signaling Technology), p38(1:200, Santa Cruz Biotechnology Biotechnology, Dallas, TX, USA), Phospho-p38(p-p38, 1:1000, Cell Signaling Technology), PEDF(1:500, Abcam), VEGF(1:1000, Abcam) and HRP-conjugated anti-β-actin antibody (1:20,000, Sigma, Saint Louis, MO, USA) as a loading control. After washing each membrane three times with Tris-buffered saline containing 0.05% Tween 20 for 10 min, they were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, Sigma) or HRP-conjugated goat anti-mouse IgG (1:10,000, Sigma) for 1 h at room temperature. The specific bands were visualized by an enhanced chemiluminescence reagent (ECL, Lulong Inc, Xiamen, China), and the image intensity was calculated with a transilluminator (ChemiDoc XRS System; Bio-Rad, Philadelphia, PA, USA).

For protein extraction, the cells were sonicated on ice in RIPA Buffer (Biosesang, Seongnam, Korea) containing a protease inhibitor cocktail. After centrifugation at 12,000 rpm at 4°C for 20 min, clear cell lysates were measured for protein concentration by Bradford assay. A total of 20 μg protein was fractionated by SDS-PAGE on 8–16% Tris- glycine gel (Komabiotech, Seoul, Korea), transferred to nitrocellulose membrane (Invitrogen), and then blotted with antibodies against LC3 II (1:500), p62/SQSTM1 (1:4000), beclin 1 (1:10000), ATG5 (1:500), Nrf2 (1:1000) (Novus biological, Littleton, USA), pro-IL-1β (1:1000), p38 (1:1000), phospho-p38 (pp38, 1:1000) (Cell Signaling Technology, Danvers, MA), or β–actin (1:200, Santa Cruz Biotechnology).