P mkk7

VSMCs were harvested on ice‐cold lysis buffer. In some experiments, VSMCs were infected with adenovirus encoding Hic‐5 (Ad‐hic‐5/flag) or β‐galactosidase (Ad‐β‐gal) as a control. Aortic tissue samples were crushed with a biomasher (Nippi) and lysed in lysis buffer. Equal amounts of protein were separated by 10% SDS‐PAGE. Immunodetection was performed using the following primary antibodies: Hic‐5; GAPDH and MMP2 (Santa Cruz Biotechnology), membrane type 1 (MT1)‐MMP (Sigma‐Aldrich); extracellular signal‐regulated kinase (ERK), p38, mitogen‐activated protein kinase kinase (MAPKK; also known as MKK) 4, MKK7, and their phosphorylated (P) forms (P‐ERK, P‐p38, P‐MKK4, P‐MKK7, and P‐JNK1/2) (Cell Signaling); and JNK1/2 (Santa Cruz Biotechnology). The densities of the bands were measured using Light‐Capture and Densitograph software (AE‐6962FC, CS Analyzer version 2.0; ATTO).

Primary antibodies against Nrf2, NQO1, β-actin, and Lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); HO-1, phospho (p)-ERK1/2, p-JNK, p-p38, p-Akt, p-MAPK/ERK kinase (MEK)1/2, p-MKK3/MKK6, p-B-Raf, mixed lineage protein kinase (MLK)3, stress-activated protein kinase/ERK kinase-1 (SEK1)/MKK4, p-MKK7, phospho-apoptosis signal-regulating kinase (p-ASK)1, and phospho-transforming growth factor β-activated kinase (p-TAK1) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Peroxidase-conjugated secondary anti-rabbit and anti-goat antibodies were purchased from Santa Cruz Biotechnology, and Alexa-Fluor-555-conjugated secondary antibody was from Cell Signaling Technology (Boston, MA, USA). Specific inhibitors including U0126 (MEK1/2 inhibitor), SP600126 (JNK inhibitor), SB202190 (p38 MAPK inhibitor), and LY294002 (PI3 kinase inhibitor) were obtained from Cell Signaling Technology. Triton X-100, polyethylene glycol and other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).