Anti pp42 44

Immunoblotting and immunoprecipitation were performed as previously described^{52 (link)}. Anti-MSK1, anti-Aurora B, anti-β-actin, anti-NFATc2, anti-GADPH, anti-α-tubulin, anti-laminA/C were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-H3S10 and anti-H3 were from Active Motif (Carlsbad, CA, USA). Anti-STAT3, anti-AKT, anti-p42/44, anti-p-STAT3 Y705, anti-p-STAT3 S727, anti-p-AKT S483, anti-p-p42/44 were from Cell Signaling Technology (Danvers, MA, USA).

SCC13 TLR4 overexpressing and SCC13 control cells were grown in 6 cm dishes in serum free DMEM medium for 24 hours. Afterwards cells were stimulated with ultrapure LPS (10μg/ml, Sigma) for different time intervals (0–60 min). Cells were lysed in RIPA buffer; the lysates were collected after each time interval and subjected to SDS-PAGE, followed by immunoblotting. The phosphorylated and non-phosphorylated form of ERK1/2 and JNK was detected by using specific rabbit polyclonal antibodies (anti-pp42/44 (#9101), anti-p42/44 (#9102); anti-rabbit HRP (#7074), all from Cell Signaling; anti-β-Actin (sc-47778, Santa Cruz Biotechnology, anti-mouse HRP (ab6728, Abcam). The expression of MyD88 (ab2064, Abcam) and IRAK-1 (D51G7, #4504, Cell signaling) upon LPS stimulation was analyzed using the same protein lysates. The secondary HRP-conjugated antibodies were used based on their species specificity to the primary antibodies.
ELISA: Cells were grown in 24 well plates till they reach 60% density. The expression of secreted IL-6 was analysed by ELISA (R&D Systems) before and after LPS stimulation at 6h and 24 hours. As controls unstimulated TLR4+ and TLR4- cells were used.

Cells were lysed in JS buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 5 mM EGTA, 1 mM Na₃VO₄, and 1X protease inhibitor cocktail). Protein concentration was determined with the Bradford assay (BioRad, Milan, Italy) using bovine serum albumin as the standard, and equal amounts of proteins were analyzed by SDS-PAGE (12% acrylamide). Gels were electroblotted into nitrocellulose membranes (G & E Healthcare, Milan, Italy). Membranes were blocked for 1 h with 5% non-fat dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20, and incubated at 4°C overnight with the primary antibody. Detection was performed by peroxidase-conjugated secondary antibodies, using the enhanced chemiluminescence system (Thermo Euroclone, Milan, Italy). Primary antibodies used were: anti-NRAS, anti-ERK1, anti-SKP2, anti-ROCK1 (Santa Cruz Biotechnologies, MA, USA), anti-pP42/44, anti-pAKT, anti-AKT, anti-pmTOR, anti-mTOR, anti-pGSK3β, anti-GSK3β, anti-pELK, anti-ELK (Cell Signaling, Danvers, MA, USA), and anti-βactin (Sigma Aldrich, Milan, Italy).