Anti p erk

Total proteins were extracted from heart tissues or RAW264.7 cell lysates and supplemented with a cocktail of protease inhibitors and phosphatase inhibitors. Proteins were quantified to an equivalent amount, separated by SDS-PAGE gel, and electro-transferred to a PVDF membrane. Having been blocked in 5% skimmed milk, membranes were incubated with antibodies: anti-LFA-1 (ITGAL, Affinity Biosciences. DF5625, 1:1000), anti-p-JNK (Affinity Biosciences. AF3318, 1:1000), anti-total JNK (Affinity Biosciences. AF6138, 1:1000), anti-p-ERK (Affinity Biosciences. AF1015, 1:1000), anti-total ERK (Affinity Biosciences. AF0155, 1:1000), anti-p-p38(Affinity Biosciences. AF4001, 1:1000), anti-total p38 (Affinity Biosciences. AF6456, 1:1000) and GAPDH. The loading control was GAPDH (Proteintech, 60004-1-lg, 1:10000). Western blot bands were analyzed with the ChemiDicTM XRS + Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

Proteins were subjected to SDS-PAGE on polyacrylamide gels (8–10%) and transferred onto a PVDF membrane. After blocking with 5% non-fat milk in TBS containing 0.1% Tween-20, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: anti-p-NF-κB P65, anti-NF-κB P65, anti-p-mTOR, anti-p-ERK, anti-ERK, anti-PDCD4, anti-GNA12 (Affinity, USA); anti-mTOR, anti-GAPDH, anti-TSG101, anti-CD63, anti-CD81, anti-Bax (Proteintech, USA); anti-Bcl-2 (Abclonal, China). Subsequently, the peroxidase-conjugated AffiniPure goat anti-rabbit or mouse IgG (Proteintech, USA) was added. Bound antibody was visualized via ECL plus TM Western blotting system detection kit (Amersham, USA).

The PAs homogenate tissue and collected HPASMCs and HPAECs were dissolved in proteolytic buffer for 30 minutes. After 30 minutes, the lysed PAs homogenate tissue and cell proteins were centrifuged at 13 500 g for 15 minutes, and then the protein concentration was measured using a BCA kit. Thirty micrograms of cell lysate from each sample were used for SDS‐PAGE (Bio‐Rad Laboratories), and Western blotting were analysed according to the protocol as described previously.²³ The chosen antibodies included anti‐NDUFA4L2 (Catalogue number: 16480‐1‐AP; ProteinTech), anti‐HIF1α (Catalogue number: AF1009; Affinity), anti‐PCNA (Catalogue number: 10205‐2‐AP; ProteinTech), anti‐cyclin A (Catalogue number: 13295‐1‐AP; ProteinTech), anti‐cyclin E (Catalogue number: 11554‐1‐AP; ProteinTech), anti‐ERK (Catalogue number: 16443‐1‐AP; ProteinTech), anti‐p‐ERK (Catalogue number: AF1015; Affinity), anti‐5‐LO (Catalogue number: AF4699; Affinity), anti‐p‐5‐LO (Catalogue number: AF8359; Affinity), anti‐p38 (Catalogue number: 14064‐1‐AP; ProteinTech), anti‐p‐p38 (Catalogue number: 4511T; Cell Signaling), anti‐JNK (Catalogue number: 51151‐1‐AP; ProteinTech) and anti‐p‐JNK (Catalogue number: 4668S; Cell Signaling) et al. After extensive washes membranes with TBS‐T, the ECL luminescent solution is added for exposure and development.