P mkk3 mkk6

After ischemia-reperfusion injury, the cultured cells, or sliced brain tissue from infracted hemisphere, were dissected and homogenized in lysis buffer. The protein extracts were separated on SDS-polyacrylamide gel and electroblotted onto polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4℃ in a buffer with 0.1% Tween with primary antibodies specific to GLP-1 (Abcam, Cambridge, MA, USA), GLP-1R (Abcam), IB-1 (BD Transduction Laboratories, San Diego, CA, USA), phospho-SAPK/JNK (Cell Signaling Technology, Beverly, MA, USA), SAPK/JNK (Cell Signaling Technology), phospho-c-Jun (Cell Signaling Technology), p-MKK3/MKK6 (Cell Signaling Technology), phospho-p38 MAPK (Cell Signaling Technology), p38 (Cell Signaling Technology), p44/42 MAPK (Cell Signaling Technology), MMP-9 (Millipore), COX-2 (Cayman, Ann Arbor, MI, USA), β-actin (Sigma-Aldrich), and Transcription factor IID (TFIID) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following this, membranes were then incubated with appropriate secondary antibodies. The signal was then detected using a chemiluminescent kit (Thermo Scientific, Rockford, IL, USA). Signals were normalized with β-actin or TFIID to standardize equal protein loading.

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).