Human phospho mapk array

HEK293 mH_XR cells were seeded in 6-well plates at 1×10⁶ cells/well and cultured for 24 h. After incubation with or without 10 µM HA for 5 min, cells were analyzed using the MAPK array according to the manufacturer's protocol (Human Phospho-MAPK-Array; R&D System, Minneapolis, MN, USA [36] (link)). Protein concentrations of cellular lysates were determined by Bradford protein assay (BioRad Laboratories) and 300 µg protein were subjected to each membrane. For quantitative analysis, the pixel density of every individual spot was determined and intensities of the spots without HA-stimulation were subtracted from the intensities of the corresponding spots with HA-stimulation.

Phosphorylation status of mitogen-activated protein kinases (MAPKs), extracellular signal regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs) and p38 isoforms were evaluated using the Human Phospho-MAPK Array (R&D Systems Europe, UK) as per the manufacturer’s instructions. The ERK inhibitor (FR180204) and Atk inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate) were purchased from CalbioChem (Merck KGaA, Germany) and used at IC₅₀ = 10 μM, a concentration previously determined to offer optimal specific inhibition relative to off target effects.

Phosphorylation status of MAPKs (mitogen-activated protein kinases), extracellular signal regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNKs) and p38 isoforms were evaluated using the Human Phospho-MAPK Array (R&D Systems) as per the manufacturer's instructions. The ERK inhibitor (FR180204) was purchased from CalbioChem (Merck KGaA, Germany) and used at IC₅₀=10 μM, a concentration previously determined to offer optimal specific inhibition relative to off target effects, which was used previously in our laboratory52 (link).
Phosphorylation of NF-κB p65 was assessed using the InstantOne ELISA in cell lysates from treated and untreated tenocytes. Absorbance was measured at 450 nm by microplate reader with positive and negative controls supplied by the manufacturer. The relative absorbance of stimulated versus unstimulated cells was used to assess the total or phosphorylated NF-κB p65 in each sample. The NF-κB inhibitor (SN50) was purchased from CalbioChem (Merck KGaA, Germany) and used at IC₅₀=11 nM.

LCLs were seeded at a density of 2.5 × 10⁶ cells in 6-wells plates containing RPMI-1640 supplemented with 0.5% FBS and incubated for 24 hours at 37 °C, in order to synchronize cell growth. Subsequently, 9.5% FBS was added to each well and cells were incubated in the presence or in the absence of 10 ng/ml of human recombinant IL-6 (R&D systems, Minneapolis, MN) for 15 min. Cells were then lysed and total proteins were extracted in the presence of Complete EDTA-free Protease Inhibitor Cocktail Tablets (Roche, Penzberg, Germany). According to the manufacturer’s protocol, 200 μg cell lysate was incubated with each human phospho-MAPK array (R&D Systems, Minneapolis, MN). Cell lysates were diluted and incubated overnight with nitrocellulose membranes in which capture and control antibodies against 43 different kinases and transcription factors, have been spotted in duplicate. The arrays were washed to remove unbound proteins and were incubated with a cocktail of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied and a signal was produced at each capture spot corresponding to the amount of phosphorylated protein bound. Pixel densities on developed X-ray film were collected and analyzed using a transmission mode scanner and Digimizer image analysis software (MedCalc software, Ostend, Belgium).

mEGFP-HRas, mEGFP-HRasG12V and mRFP-RBD(R59A)-mRFP plasmids were a gift from Karel Svoboda (Addgene plasmids respectively: #18662, #18666, #18664) [46 (link)]. FLAG-MPP1 for MPP1 recomplementation was described before [27 (link)]. Anti-MPP1 antibodies were purchased from Abnova, anti-GAPDH (Abcam), anti-FLAG (Sigma-Aldrich), anti-P-IGFIRβ(Tyr1135/1136)/IRβ(Tyr1150/1151), anti-IRβ, anti-P-Erk1/2(Thr202/Tyr204), anti-Erk1/2, anti-P-Akt(Ser473), anti-Akt, anti-P-p38(Thr180/Tyr182) and active Ras Detection Kit (Cell Signaling Technology), anti-HRas antibodies (GeneTex), anti-N and KRas antibodies (Aviva Systems Biology), Human Phospho-MAPK Array (R&D Systems), anti-GFP, Secondary antibodies conjugated with HRP (SantaCruz), Cholera Toxin β conjugated with HRP (Life Technologies).