Anti p44 42 erk1 2

The antimicrobial peptides hBD-1, hBD-2, hBD-3, hBD-4, and HNP-4 were obtained from the Peptide Institute (Osaka, Japan) and dissolved in 0.01% acetic acid. LL-37 (L¹LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES³⁷) was synthesized by the solid-phase method, as previously reported [24 (link)]. AG1478 was purchased from Selleck (Houston, TX). PP2, JNK inhibitor II, U0126, SB203580, and NF-κB activation inhibitor II were obtained from Calbiochem (La Jolla, CA, USA). Can Get Signal^® Immunoreaction Enhancer Solution (TOYOBO Co., Ltd., Osaka, Japan) was used as the diluent for the antibodies.
The primary antibodies anti-EGFR (1:2000 dilution), anti-phospho-EGFR (1:1000 dilution), anti-SAPK/JNK (1:500 dilution), anti-phospho-SAPK/JNK (1:1000 dilution), anti-p38 (1:1000 dilution), anti-phospho-p38 (1:2000 dilution), anti-p44/42 (ERK1/2) (1:2000 dilution), anti-phospho-p44/42 (ERK1/2) (1:2000 dilution), and anti-NF-κB p65 (1:1000 dilution) were purchased from Cell Signaling Technology (Beverly, MA, USA), and anti-Lamin B1 (1:20,000 dilution) was obtained from Proteintech (Rosemont, IL, USA). Horseradish peroxidase (HRP)-linked anti-rabbit IgG and HRP-linked anti-mouse IgG secondary antibodies were purchased from Cytiva (Tokyo, Japan) and diluted at 1:2000.

Fifty micrograms of proteins were separated on 14% (for CCL2 detection) and 10% (for signaling pathway analysis) SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and incubated with anti-CCL2 (Thermo Fisher Scientific, Waltham, MA, USA, cat. N. MA5-17040), anti-JAK2 (Cell Signaling Technology, Danvers, MA, USA, cat. N. 3230), anti-phophoJAK2 (Tyr 1007/1008) (Cell Signaling Technology, cat. N. 3771), anti-STAT5 (Cell Signaling Technology, cat. N. 9358), anti-phosphoSTAT5 (Tyr 694) (Cell Signaling Technology, cat. N. 9314), anti-Akt (Cell Signaling Technology, cat. N. 9272), anti-phopshoAkt (Ser 473) (Cell Signaling Technology, cat. N. 9271), anti-P44/42 ERK1/2 (Cell Signaling Technology, cat. N. 4695), anti-phosphoP44/42 ERK1/2 (Thr202/Tyr 204) (Cell Signaling Technology, cat. N. 9101) and anti-GAPDH (Merck Millipore, Burlington, MA, USA, cat. N. MAB374). Data are expressed as relative western blot optical density (O.D.) values using GAPDH as loading control protein. For specific calculations, please refer to the figures’ legends.

For western blotting, cells were lysed using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce, Rockford, IL, USA) containing a PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche), an EDTA-free protease tablet and 10 μl (25 units/μl) benzonase (70746-4 Novagen®, Merck Millipore, Darmstadt, Germany). Transfected HEK293T cells were lysed for 20′ at 4°C and centrifugated at 2000 g at 4°C to obtain protein lysates.
Proteins were electrophoretically separated and blotted onto a nitrocellulose membrane (Whatman, Kent, UK). This membrane was blocked for 1 h in 5% non-fat dry milk and afterwards incubated overnight (4°C) in one of the following primary antibodies: anti-phospho-SAPK/JNK (Thr183/Tyr185, #4668), anti-phospho-p44/42 (ERK1/2, #4370), anti-SAPK/JNK (#9252), anti-p44/42 (ERK1/2, #9102) (Cell Signaling Technologies, MA, USA), or anti-β-Actin (A5316, Sigma Aldrich, MO, USA). After washing, the membranes were incubated with either a secondary goat anti-rabbit (ab6721, Abcam, Cambridge, UK) or sheep anti-mouse (NA931, GE Healthcare, Buckingmersham, UK) antibody. Finally, the corresponding proteins were visualized using Enhanced ChemiLuminescence Western Blotting Substrate (Thermo Scientific, IL, USA).

MCs were starved overnight in RPMI 1640 supplemented with 5%FCS and 15 ng/ml recombinant murine IL-3 and then stimulated with IL-33 (100 ng/ml) and/or SCF (100 ng/ml) alone or in combination for 15 min at 37 °C. Cells were homogenized in fresh prepared RIPA lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, Triton X-100 1%, SDS 0.1%, DOC 0.5%, EDTA 1 mM, EGTA 1 mM, MgCl2 5 mM, leupeptin 5μg/ml, PMSF 1 mM, aprotinin 5μg/ml, NaF 5 mM, Na₃VO₄ 1 mM). Lysates were centrifugated at 13,000 g for 20 min to eliminate debris, resolved by SDS-polyacrylamide gel (PAGE) and proteins were electro-blotted, as previously described [70 (link)].
Immunoreactive bands were visualized on the nitrocellulose membranes, using horseradish-peroxidase-linked/coupled donkey anti-rabbit or sheep anti-mouse IgG (GE Healthcare, Chicago, IL) and the Clarity max, western ECL substrate (Biorad, Hercules, CA) based on manufacturer’s instructions and exposed to Invitrogen iBrightTM Imaging Systems (Thermo Scientific).
The following Abs were used: anti-pSTAT3, anti-STAT3, anti-pAkt S473/D9E, anti-Akt, anti-pp38 T880/Y182, anti-p38, anti-pp44/42 (ERK1/2) (T202/Y204) and anti-P44/42 (ERK1/2) (all from Cell Signaling, Danvers, MA); anti-Ikb-α (Santa Cruz Biotechnologies, Dallas, TX); anti-actin (Merck Life Sciences). Densitometric analysis was performed using FIJI Image J software.