Rabbit anti phospho p44 42 mapk

Total protein from hGSMCs was separated using SDS-PAGE and transferred on a PVDF membrane. Membranes were incubated overnight at 4 °C with primary antibodies: rabbit anti-phospho-p44/42 MAPK (1:1000, #9101, Cell signalling) and rabbit anti-p44/42 MAPK (1:1000, #9101, Cell signalling) or mouse anti-vinculin (1:10000, Sigma-Aldrich) as a protein-loading control. Secondary antibodies used were peroxidase conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:5000, Sigma-Aldrich) (1h, RT). Bands were quantified by relative densitometry and normalized to vinculin, using ImageQuant TL 8.1 (GE Healthcare).

Cell lysates were homogenized in lyses buffer (Beyotime, China) and protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The analysis of protein was performed according to standard SDS–PAGE. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) plus detection reagent (Pierce, Rockford, IL) and analyzed using an Omega 16ic Chemiluminescence Imaging System (Ultra-Lum, CA). The following primary antibodies were used: rabbit anti-Phospho-p38 MAPK (4511, Cell Signaling Technology, USA), rabbit anti-p38 MAPK (8690, Cell Signaling Technology, USA), rabbit anti-Phospho-p44/42 MAPK (4370, Cell Signaling Technology, USA), rabbit anti-p44/42 MAPK (4695, Cell Signaling Technology, USA), rabbit anti-Phospho-SAPK/JNK (4668, Cell Signaling Technology, USA), rabbit anti-SAPK/JNK (9252, Cell Signaling Technology, USA), rabbit anti-Phospho-IKKα/β (2697, Cell Signaling Technology, USA), rabbit anti-IKKβ (8943, Cell Signaling Technology, USA), rabbit anti-Phospho-NF-κB p65 (3033, Cell Signaling Technology, USA), rabbit anti-NF-κB p65 (8242, Cell Signaling Technology, USA).

Whole-liver lysates were prepared in Triton-X 100 lysis buffer containing protease inhibitors and protein concentrations were quantified using the Bradford method with bovine serum albumin (BSA) as the standard. Resolution by SDS-PAGE was followed by immunoblotting using the following antibodies: rabbit anti-PDGFRα (#3164), rabbit anti-phospho-p44/42 MAPK (#9101), rabbit anti-p44/42 MAPK (#9102), rabbit anti-β actin (#4(a967), all from Cell Signaling (Danvers, MA) and anti-phospho-Smad3 (#ab52903) from Abcam (Cambridge, MA). Epitope-primary antibody complexes were detected using species-specific secondary antibodies conjugated to horseradish peroxidase (HRP) followed by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Pierce, IL).

The protein samples obtained from tissue or cell extracts were quantified using the Bradford reagent (BioRad). Before loading into the 10% SDS-PAGE, the samples were mixed with 5x loading buffer and boiled at 98 °C for 5 mins. After gel electrophoresis, the proteins were then transferred to a PVDF membrane (0.45 μm, Merck Millipore) under 250 mA for 90 mins. The membranes were incubated with a blocking buffer (5% nonfat milk in Tris-buffered saline containing 0.5% Tween-20) for 1hr at room temperature and then incubated with the indicated primary antibodies at 4 °C overnight. The membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG; 1:5000) or with goat anti-rabbit IgG (1:5000) for one hour at room temperature. The target protein bands were visualized using Amersham Imager 600 (GE healthcare). The following antibodies were used: goat Anti-Serotonin transporter (Abcam, #ab130130), rabbit anti-p44/42 MAPK (Cell Signaling, #4695 s), rabbit anti-Phospho-p44/42 MAPK (Cell Signaling, #4370 s), mouse anti-ß-actin (Beijing TDY Biotech LTD, #M009), HRP-conjugated secondary mouse (Beijing TDY Biotech LTD, #E009) goat (Beijing TDY Biotech LTD, S008) and rabbit antibodies (Beijing TDY Biotech LTD, #E011).

Sphingosine-1-phosphate, W146, (4R)-2-Undecyl-4-thiazolidinecarboxylic acid (BML-241), FITC-phalloidin, L-alpha-phosphatidylcholine palmitoyl and fatty acid free bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, USA). Rabbit anti-phospho-Akt (catalog number: 9271), rabbit anti-Akt (catalog number: 4685), rabbit anti-p44/42 MAPK (catalog number: 4695), rabbit anti-phospho-p44/42 MAPK (catalog number: 9101) and goat anti-rabbit HRP-linked (catalog number: 7071) polyclonal or monoclonal antibodies were purchased from Cell Signaling (Danvers, USA), whereas the mouse anti-actin monoclonal antibody (clone C4—catalog number: MAB1501) was from Merck Milipore (Darmstadt, Germany) and the goat anti-mouse HRP-linked antibody (catalog number: 1031–05) was from Southern Biotech (Birmingham, USA).

Immunoblotting was used to measure apoptosis and adiponectin expression following a standard Bio‐Rad protocol. The primary antibodies used were 1:1000 rabbit anti‐cleaved PARP (Cell Signaling 5625), 1:1000 rabbit anti‐cleaved caspase‐3 (Cell Signaling 9664), 1:1000 rabbit anti‐adiponectin (Abcam, Cambridge, UK; ab3455), 1:1000 rabbit anti‐phospho‐c‐Jun N‐terminal kinases (p‐JNK; Thr183/Tyr185) (Cell Signaling 9251), 1:1000 rabbit anti‐total JNK (T‐JNK) (Cell Signaling 9252), 1:1000 rabbit anti‐phospho‐p38 mitogen‐activated protein kinases (p‐p38 MAPK; Thr180/Tyr182) (Cell Signaling 4092), 1:1000 rabbit anti‐total p38 MAPK (T‐p38 MAPK) (Cell Signaling 8690), 1:1000 rabbit anti‐phospho‐p44/42 MAPK (phospho‐extracellular signal‐related kinases 1 and 2 [p‐ERK1/2]; Thr202/Tyr204) (Cell Signaling 9101), 1:1000 rabbit anti‐total p44/42 MAPK (T‐ERK1/2) (Cell Signaling 9102), and 1:10,000 mouse anti‐β actin (Sigma A5316). All secondary antibodies were purchased from Cell Signaling. An ELISA was used to measure adiponectin (R&D Systems, Minneapolis, MN, USA; MRP300) and TNF‐α (R&D Systems MHSTA50) according to the manufacturer's instructions.