Rabbit anti p44 42 mapk

Kinase activation for ERK1/2 was determined by measuring the level of phosphorylated kinase and normalizing the value to the total protein level of ERK1/2. Phospho- and total-ERK1/2 were detected and analyzed by LI-COR odyssey Fc dual-mode western blot system. Mouse anti-phospho-p44/42 MAPK (T202/Y204) (Cell signaling) and donkey anti-mouse IRDye 800CW (LI-COR) were used to detect activation of ERK1/2 by green fluorescence channel. Rabbit anti-p44/42 MAPK (Cell signaling) and goat anti-rabbit IRDye 680RD (LI-COR) were used to detect total ERK1/2 by red fluorescence channel.

Cells were harvested and lysed in the following lysis buffers: for unprocessed Rap1 detection, boiling hot lysis buffer containing 100 mM Tris-HCl, 1.1% SDS, 11% glycerol was used; for the other proteins, lysis buffer containing 50 mM Tris-HCl, 1% SDS, 1 mM DTT and 0.43 mM ABSF was used. After then, lysates of cells were sonicated and centrifuged at 20,400 g for 20 min at 4°C, and the supernatant was collected. Equal amounts of the protein extract were subjected to SDS-PAGE, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The following were used as the primary antibodies: rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-p44/42 MAPK (ERK1/2), rabbit anti-phospho-p44/42 MAPK (ERK1/2), rabbit anti-PARP (Cell signaling Technology, Beverly, MA, USA), mouse anti-β-actin (Sigma-Aldrich), rabbit anti-BIM (Abcam, Cambridge, UK), mouse anti-TRAIL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-HMGCR (Abcam) and mouse anti-Rap1 (Santa Cruz Biotechnology). The signals were detected with a Chemi-Lumi One L (Nacalai Tesque) or an Immobilon™ Western Chemiluminescent HRP Substrate (Millipore).

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).

Total protein extracts were prepared as described (Kushnirov, 2000 (link); von der Haar, 2007 (link)) with subtle modifications. Briefly, 8 ml of mid-log phase cells were treated with 10% trichloroacetic acid and pelleted by centrifugation. Cell pellets were washed with 10 ml 70% ethanol and 2x with 1 ml water, then re-suspended in 1 ml 0.2 M NaOH and incubated 10 min on ice. Cells were pelleted, resuspended in 160 x OD₆₀₀ μl loading dye (120 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 0.02% bromophenol blue, 20% glycerol), heated at 95°C for 10 min, and lysates clarified by centrifugation at 16,000 x g. Proteins were separated on 10% tris-glycine SDS-PAGE gels, transferred to 0.45 μm nitrocellulose membranes (Bio-Rad) and probed overnight at 4°C with mouse anti-HA (1:5,000; Sigma-Aldrich, 12CA5), rabbit anti-PSTAIR (1:5,000; Millipore-Sigma, 06–923), rabbit anti-p44/42 MAPK (1:2,500; Cell Signaling Technology, 9102) or rabbit anti-G6PDH (1:5,000; Sigma-Aldrich, A9521). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were from Jackson ImmunoResearch (115–035-003 or 111–035-003) and used at 1:10,000 dilution for 60 min at 4°C. Immunoblots were developed using Clarity Western ECL Substrate (Bio-Rad) and imaged on a ChemiDoc MP multimode imager (Bio-Rad).

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).