2.11. Western blot analysis

Western blot analysis was performed to examine Nrf2 knockdown efficiency using double transfection of KC with Nrf2-siRNA and detect phosphorylated MAPKs (ERK, JNK, and p38) in MC. Total protein extracts were prepared as previously described [16]. The membranes were blocked in 5% (w/v) skim milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 for 1.5 h and incubated at 4 °C overnight with the primary antibody against Nrf2 (sc-722; Santa Cruz Biotechnology, Santa Cruz, CA) (1:2000), phosphorylated Erk1/2 (p44/42 MAPK (Thr202/Tyr204) (4370S; Cell Signaling) (1:2000), phosphorylated JNK (Thr183/Tyr185) (G9) (9255S; Cell Signaling) (1:1000), phosphorylated p38 (Thr180/Tyr182) (D3F9) (4511S; Cell Signaling) (1:1000), p65 (Sc-109; Santa Cruz Biotecnology) (1:1000) and p50 (Sc-114; Santa Cruz Biotecnology) (1:1000) subunits of NF-κB, phospho-p53 (Ser15) (9284; Cell Signaling) (1:1000) and p53 (2524; Cell Signaling) (1:1000), in 5% skim milk. The membranes were washed with a PBS solution and incubated for 1.5 h at room temperature with the HRP-conjugated secondary antibodies (ab6789 for anti-mouse and ab6721 for anti-rabbit HRP labelled secondary antibody; Abcam, Cambridge, MA, USA) (1:2000) in 5% skim milk. Immunoreactivity is detected using the Bio-Rad Clarity western ECL (Bio-Rad). Protein bands were visualized using an ImageQuant LAS 4000 digital imaging system (GE Healthcare, UK) and analyzed as previously described [16]. The protein expressions were normalized to expression of loading controls; α-Tubulin (ab7291; Abcam, Cambridge, MA, USA) (1:5000) for Nrf2 protein in whole cell lysates, total ERK protein (p44/42 MAPK) (Erk1/2) (4695 S; Cell Signaling) for phosphorylated ERK, total JNK protein (9252S; Cell Signaling) for phosphorylated JNK, total p38 protein (9212S; Cell Signaling) (1:2000) for phosphorylated p38, βactin (A3854; Sigma) (1:10000) for cytosolic p65 and p50 subunits of NF-κB and phospho-p53 (Ser15) and lamin A (sc-20680, Santa Cruz Biotecnology) (1:1000) for nuclear p50 and p65 subunits of NF-κB.