Anti rabbit ig

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).

Immunoblotting was performed with a standard protocol^{5 (link)}. We used primary antibodies against pSIKK1/2/ IKK1/2, pT/Y-p38/ p38, anti-MK2, anti-MK3, pJNK1/2/ JNK1/2 and JNK1 (all Cell Signaling except anti-IKK1/2 and tubulin which were from Santa Cruz) and secondary antibodies conjugated with HRP [anti-rabbit-Ig, anti-goat-Ig (both Santa Cruz) and anti-mouse-Ig (Thermo Fisher Scientific)]. Detection was performed using ECL reagent (Pierce). Western blots were digitally developed with the ImageQuant 4000 system (GE Healthcare Life Science, England) or with X-Ray films (Fuji).

RIPA lysate (P0013B/; Beyotime, Shanghai, China) was used to lyse the cells and extract proteins. A BCA kit (P0010S; Beyotime, Shanghai, China) was used to measure the protein concentration. 10% SDS-PAGE gel electrophoresis and PVDF membrane (Millipore, IPVH00010) were used to separate and transfer protein samples (about 20 µg), respectively. The PVDF membrane was incubated with skim milk at 25 °C for 1 h, and then incubated with the first antibodies (Anti-GAPDH: 1:2,000, sc-32233, Santa-Cruz; Anti-FBXO43: 1:500, HPA024292, Sigma) at 4 °C for the night. The membrane was washed with TBST solution (with 0.1% TWEEN-20). Then, the secondary antibody (Anti-Rabbit-Ig: 1:10000, sc-2004, Santa Cruz; Anti-mouse IgG: 1:2000, sc-2005, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to incubate the membrane at 25 °C for 2 h. The membrane was washed with TBST solution. Finally, the protein bands were detected by using a ECL-PLUS/Kit (M3121; Thermo Fisher, Waltham, MA, USA) in a gel imaging system.