Sc 66163

Cerebral cortex was lysed using RIPA buffer with a proteinase inhibitor. The samples were diluted to the same quantities (20 μg) and then loaded. Protein samples were separated by SDS-PAGE electrophoresis on 8–10% gels and then electro-transferred onto PVDF membranes (Millipore Corp). The membranes were blocked and then incubated with primary antibody (1:3,000) overnight at 4°C. Subsequently, the membrane was incubated with secondary antibodies (1:3,000 Abcam, anti-mouse IgG or anti-rabbit IgG). Then the blot was detected using the Western Bright ECL western blotting detection kit (Bio-Rad Laboratories). Equal sample loading was verified by the detection of GAPDH. The primary antibodies were as follows: mouse monoclonal anti-GAPDH (sc-66163, Santa Cruz, CA, United States), rabbit monoclonal anti-Fyn (ab125016, Abcam), rabbit monoclonal anti-SRPK2 (ab192238, Abcam), mouse monoclonal anti-tau (sc-390476, Santa Cruz, CA, United States), mouse monoclonal anti-tau (phospho Tyr18, 9G3, Genetex), and rabbit monoclonal anti- tau (phospho Ser214, ab170892, Abcam).

Proteins were extracted from the cardiac fibroblasts and myocardial tissues homogenized in RIPA Lysis (P0013B; Beyotime) and Extraction Buffer with a protease inhibitor cocktail, and proteins were quantified using the bicinchoninic acid method. Samples of 25-μg protein were loaded into 8% SDS-PAGE gels for electrophoresis then transferred to PVDF membranes overnight at 30V. Antibodies specific for α-SMA (dilution 1:500; ab5694; Abcam), YAP (dilution 1:100; 4912; Cell Signaling), p-YAP (dilution 1:100; 4911; Cell Signaling), and collagen I (dilution 1:1000; ab93095; Abcam) were incubated at 4°C overnight, and GAPDH (dilution 1:5000; sc66163; Santa Cruz, USA) was used as a loading control to normalize gel loading and protein expression. HRP-conjugated secondary antibodies (dilution 1:300; AS10 653; Agrisera, Sweden) plus ECL (AS16; Agrisera) were incubated at 37°C for 1 h for protein visualization. The densitometric values of bands were measured using densitometry analysis software (Multi Gauge Ver 3.0, Japan).