Anti phospho iκbα ser32 36 5a5

Western blotting was performed as previously described (Chen et al., 2002 (link); Xiao et al., 2019 (link)). Antibodies anti-myeloid differentiation primary response gene 88 (MyD88) (D80F5; #4283), anti-phospho-IKKα/β (Ser176/180) (16A6; #2697), anti-IKKα (#2682), Phospho-IKKα/β (Ser176/180) (16A6, #2697), anti-IκBα (44D4,#4812), anti-Phospho-IκBα (Ser32/36) (5A5, #9246), anti-NF-κB p65 (D14E12, #8242) and anti-Phospho-NF-κB p65 (Ser536) (93H1, #3033) were purchased from Cell Signaling Technology.

Whole-cell (30 µg), cytoplasmic (30 µg) and nuclear (10 µg) lysates were resolved by SDS-PAGE and analyzed by immunoblotting. The following antibodies were used: anti-NF-κB2 p52 (C-5) (Santa Cruz Biotechnology, #sc-7386) for detection of p52 and its precursor p100; anti-NIK (Cell Signaling, Danvers, MA, USA, #4994); anti-RelB (C-19) (Santa Cruz Biotechnology, #sc-226); anti-phospho-p100 (Ser866/870) (Cell Signaling, #4810); anti-phospho-IκBα (Ser32/36) (5A5) (Cell Signaling, #5205); anti-IκBα (C-21) (Santa Cruz Biotechnology, #sc-371); anti-phospho-IKKα(Ser180)/IKKβ (Ser181) (Cell Signaling, #2681), anti-IKKα (H-744, Santa Cruz Biotechnology, #sc-7218), anti-IKKα/β (H-470, Santa Cruz Biotechnology, #sc-7607), anti-TRAF2 (C90-481) (BD Biosciences, San Jose, CA, USA, 558890); anti-TRAF3 (H-122) (Santa Cruz Biotechnology, SC-1828); anti-cIAP1 (R&D systems, Minneapolis, MN, USA, AF8181); anti-lamin A/C (4C11) (Cell signaling, #4774); anti-α-tubulin (Sigma-Aldrich, St Louis, MO, USA, T9026). For detection of endogenous NIK protein, cells were treated with 0.1% DMSO or 20 µM of MG132 (PEPTIDE INSTITUTE, Osaka, Japan) for 6 hours and cytoplasmic extracts were subjected to immunoblot analysis.