Anti ikk1 2

Samples were separated in 10% sodium dodecyl sulfate (SDS)-Laemmli gels and transferred by electroblotting onto nitrocellulose membranes (biostep). Membranes were blocked with dry milk and incubated (overnight) with antibodies detecting phosphorylated/ non-phosphorylated proteins or Hemagglutinin (HA)-tagged TAK1. We used the anti-HA antibody (provided by Prof. Böhmer, Centre of Medical Biomedicine, Jena), anti-pY719-c-Kit/c-Kit, anti-pY705-STAT3/STAT3, anti-pY694-STAT5/STAT5, anti-pS184/pS187-TAK1/TAK1, anti-pS176(IKK1)/pS-177(IKK2) or anti-pY199-IKK2 and anti-IKK1/2, anti-pS32-IκBα/IκBα, anti-pS473-PKB/Akt/PKB/Akt, anti-pY396-Lyn/Lyn, anti-Tubulin and anti-Ubiquitin [Cell Signaling; except anti-c-Kit, anti-TAK1, anti-IKK1/2, anti-Lyn and anti-Ubiquitin (Santa Cruz); anti-pY199-IKK2 and anti-pY396-Lyn (abcam) and anti-Tubulin (Sigma-Aldrich)]. Membranes were washed in 0.1% Tween/TBS and incubated with HRP-conjugated secondary antibodies: anti-rabbit-Ig, anti-goat-Ig (Santa Cruz) and anti-mouse-Ig (Thermo-scientific). Detection was performed using ECL reagent (Pierce).

Washed BMMCs were seeded in IL‐3‐free media at a density of 10⁶ cells/ml. After 1 h, cells were stimulated with rmIL‐33 (50 ng/ml) (PeproTech) and/or ATP (500 µM) (Sigma). Lysis of the cells was performed with lysis buffer (20 mM HEPES, pH7·5; 10 mM EGTA, 40 mM β‐glycerophosphate, 2·5 mM MgCl₂, 2 mM orthovanadate, 1 mM dithiothreitol, 20 µg/mL aprotinin and 20 µg/ml leupeptin supplemented with 1% Triton), and the protein concentration was determined (BCA‐kit; Pierce). Boiled samples (treated with 6 × Laemmli buffer) were separated on 10% sodium dodecyl sulphate (SDS)‐Laemmli gels. Gels were transferred onto nitrocellulose membranes (biostep) by electroblotting. After blocking (with dry milk), membranes were incubated with either anti‐pT180/pY182‐p38, anti‐pS177/181‐IKK2, anti‐p184/187‐pTAK1, anti‐pT202/pY204‐ERK1/2, anti‐pS32‐IκBα, and anti‐pS536‐p65 or the respective anti‐total antibodies (non‐phosphorylated proteins) [all from Cell Signaling except anti‐IKK1/2, anti‐ERK1/2 and anti‐tubulin (Santa Cruz)]. Washed membranes (TBS/0·1% Tween) were incubated with HRP‐conjugated secondary anti‐rabbit‐Ig, or anti‐mouse‐Ig or anti‐goat‐Ig (SeraCare). Detection was performed with ECL reagent (Pierce).

After stimulation with rmIL-33 and/or rmSCF (both 50 ng/mL) (Peprotec), BMMCs were lysed in lysis buffer (20 mM HEPES, pH 7.5; 10 mM EGTA, 40 mM β−glycerophosphate, 2.5 mM MgCl₂, 2 mM orthovanadate, 1 mM dithiothreitol, 20 µg/mL aprotinin, and 20 µg/mL leupeptin supplemented with 1% Triton). The protein concentration was determined (BCA−kit; Pierce), and the samples were boiled (with 6 × Laemmli buffer). The lysates were separated with 10% sodium dodecyl sulfate (SDS) −Laemmli gels and afterwards transferred onto nitrocellulose membranes (biostep) by Western blotting. The membranes were blocked (with dry milk) and incubated with anti-pT180/pY182−p38, anti-p38, anti-pS177/181−IKK2, anti-p184/187−pTAK1, anti-pT202/pY204−ERK1/2, anti-pS32−IκBα, anti-IκBα, anti-pS536−p65, anti-p65, HSP90, anti-pST183/Y185−JNK1/2, anti-JNK1/2, anti-pS727−STAT3, anti-STAT3, anti-pS235/236−S6, anti-S6, and anti-tubulin (all from Cell Signaling except anti-IKK1/2, anti-TAK1, and anti-ERK1/2 (Santa Cruz, CA, USA)). The membranes were washed with TBS/0.1% Tween and incubated with HRP-conjugated secondary anti-rabbit Ig, anti-mouse Ig, or anti-goat Ig (SeraCare). We used ECL reagent (Pierce) for detection.