Pde4d

Following extracting proteins from OGD/R-induced PC-12 cells by RIPA buffer containing protease inhibitors, the BCA Protein Assay Kit (Abcam) was used to detect the protein concentrations. Then, proteins were separated by 10% SDS-PAGE and transferred into PVDF membranes. At room temperature, the blocking was performed using 5% bovine serum albumin (BSA). After blocking, membranes were incubated overnight at 4°C with primary antibodies against Bcl-2 (1:1,000; Abcam), Caspase-3 (1:1,000; Abcam), PDE4D (1:1,000; Abcam), and β-actin (1:1,000; Abcam). Then, tris-buffered saline Tween-20 (TBST) was used to wash the membranes for three times. Subsequently, at room temperature, the secondary antibody HRP-conjugated anti-mouse IgG (1:5,000; Santa Cruz, Waltham, MA, USA) was added to incubate for 1 h. β-actin was used as the internal reference. The membranes were developed by chemiluminescence reagents (Thermo Fisher Scientific) under a Gel-Pro analyzer (version 4.0, USA).

Protein was extracted from the aortic tissues or RASMCs in a lysis buffer. Equal quantities of protein extract (30 μg per lane) were separated by 8, 10, or 12% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Merck, Cat#: IPVH00010). The target protein was probed with numerous antibodies: PDE4A (1:1000, Thermo Fisher, Cat#:PA5-115730), PDE4B (1:1000, Cell Signaling Technology, Cat#: 72096S), PDE4C (1:1000, Thermo Fisher, Cat#: PA5-106624), PDE4D (1:1000, Abcam, Cat#: ab171750), AMP-activated protein kinase (AMPK; 1:1000, Cell Signaling Technology, Cat#: 2532S), Phospho-AMPK (1:1000, Cell Signaling Technology, Cat#: 2535S), myosin phosphatase targeting subunit 1 (MYPT1; 1:1000, Cell Signaling Technology, Cat#: 2634S), Phospho-MYPT1 (1:500, Cell Signaling Technology, Cat#: 5163S), MLC (1:1000, Cell Signaling Technology, Cat#: 8505S), and Phospho-MLC (1:1000, Cell Signaling Technology, Cat#: 3675S), respectively. Immunoblotting of the housekeeping protein GAPDH (1:5000, Proteintech, Cat#: 60004-1-Ig) was performed to ensure equal protein loading. Immunoreactive bands were visualized with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Pierce, Cat#: 34577). The protein expression was measured by analyzing the relative protein band intensity with Image-Pro Plus 6.0 software.