Rabbit anti phospho p38

BD Matrigel™ Basement Membrane Matrix, culture dishes, and plates were obtained from Corning Inc. (Corning, NY, USA). Concanavalin A (Con A), sodium dodecyl sulphate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Endothelial Cell Medium (ECM) was obtained from ScienCell (Carlsbad, CA, USA). PD98059 and LY294002 were the products of Tocris Bioscience (Bristol, UK). The primary antibodies, such as rabbit anti-phospho-Akt (Ser473) (#4060S), rabbit anti-Akt (#9272S), rabbit anti-phospho-ERK1/2 (#4730S), rabbit anti-ERK1/2 (#4695S), rabbit anti-phospho-p38 (#4511S), rabbit anti-p38 (#8690S), mouse anti-p27 (#3686S), mouse anti-p21 (#2947S), rabbit anti-cyclin D1 (#55506S), and rabbit anti-cyclin E (#20808S) antibodies were from Cell Signalling Technology (Beverly, MA, USA). The polyvinylidene fluoride (PVDF) membranes were the products of Millipore (Billerica, MA, USA). EdU Apollo®488 In Vitro Imaging Kit was a product of RIBOBIO (Guangzhou, China).

PDLSCs were washed with ice‐cold PBS and lysed using a RIPA lysis buffer containing 1% PMSF (Solarbio) and 1% phosphatase inhibitor (Boster, Wuhan, China) and then were centrifuged at 12,000 g for 10 min. Protein concentration was measured by a BCA Protein Assay Kit. Proteins were added with SDS‐PAGE loading buffer and then denaturated at 100°C for 5 min. 20 μg/lane proteins were loaded to SDS‐PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with non‐fat dry milk for 1 hr, blotted primary antibodies overnight at 4°C and subsequently incubated with horseradish peroxidase‐labelled secondary antibodies (1:2000; Beyotime, Shanghai, China) for 1 hr at room temperature. The membrane was washed three times in tris‐buffered saline with 1‰ Tween20 (TBST). The immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore). The level of each protein was normalized to GAPDH before statistical analysis. Image J 1.44 software (NIH, Bethesda, Maryland, USA) was used to quantify the protein expression. The primary antibodies used were as follows: rabbit anti‐phospho‐ERK1/2 (1:1000; Cell Signaling Technology, Danvers MA, USA), rabbit anti‐ERK1/2 (1:2000; Cell Signaling Technology), rabbit anti‐phospho‐p38 (1:1000; Cell Signaling Technology) and GAPDH (1:10,000; Proteintech, Chicago, IN, USA).