Ab5686

The proteins were separated from the infarcted hemisphere hippocampal tissue and then separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. 10% skimmed milk or BSA was used to block for 2 h, and we incubated the membranes at 4°C overnight with anti‐MEF2D (1:1000, Abcam, Cambridge, UK, ab246884), anti‐p‐ERK5 (1:1000, Abcam, Cambridge, UK, ab5686), anti‐ERK5 (1:1000, Abcam, Cambridge, UK, ab40809) and β‐actin (1:10,000, ZSGB‐BIO, Beijing, China, TA‐09). Next, we followed this with incubation with a secondary antibody labelled by horseradish peroxidase (1:10 000, ZSGB‐BIO, Beijing, China, ZB‐2301) for 2 h. Finally, we used enhanced chemiluminescent reagent (Thermo Fisher, Waltham, MA, USA, D1306) to examine blots, and ImageJ (Rawak Software Inc, Stuttgart, Germany) to analyse blots quantitatively.

IHH4 cells were lysed using a lysis buffer containing fresh protease inhibitors (Beyotime Biotechnology, Shanghai, China). The protein concentrations of the lysate were determined using the BCA method (Beyotime). Primary antibodies (at 1:1000 dilution unless otherwise indicated) against the following proteins were used for immunoblotting assays: β-actin (#2872; CST, Beverly, MA, USA), PCNA (ab92552; Abcam, Cambridge, UK), cyclin D1 (ab134175; Abcam), cleaved caspase-3 (#29034; SAB, College Park, MD, USA), ERK1/2 (ab184699; Abcam), phospho-ERK1/2 (ab214362; Abcam), ERK5 (ab40809; Abcam), phospho-ERK5 (ab5686; Abcam), JNK (ab208035; Abcam), phospho-JNK (ab124956; Abcam), p38 (#8690; CST) and phospho-p38 (#4511; CST). HRP-conjugated goat anti-rabbit secondary IgG (1:3000) (KeyGen Biotech, Nanjing, China) were used to probe blots at room temperature for 1 h. A Tanon-4500 system (Tanon Science and Technology, Shanghai, China) was used for digital blot visualization.