Anti traf2

Western blotting experiment was described previously (Zhao et al., 2019 (link)), and the detection of RIPK3 or MLKL oligomers was performed according to the method described by Wang et al. (2019) (link). The following antibodies were used for the experiments: anti-FADD (1:1000, 610399) and anti-RIPK1 (1:3000, 610458) (BD Transduction Laboratories, San Jose, CA); anti-phospho-RIPK3 (1:1000, ab195117), anti-MLKL (1:1000, ab172868), anti-phospho-MLKL (1:1000, ab196436) (Abcam, Cambridge, MA, United States); anti-RIPK1 (1:3000, 3493), anti-RIPK3 (1:3000, 15828), anti-Myc tag (1:2000, 2276), anti-HA tag (1:2000, 3724), anti-Flag tag (1:2000, 14793), anti-TRAF2 (1:1000, 4724), anti-phospho-RIPK1 (1:1500, 31122) and anti-phospho-RIPK3 (1:1000, 57220) [Cell Signaling Technology (CST), Beverly, MA]; anti-TRADD (1:1000, ABP52634) and anti-GAPDH (1:1500, ABP52783) (Abbkine, Redlands, CA, United States); anti-β-actin (1:3000, A5441) (Sigma-Aldrich). All western blot assays were performed three times, and the representative results are shown.

Anti-phospho-TAK1 (Thr-187), rabbit anti-TAK1, anti-TRAF2, anti-TRAF6, anti-TAB1, anti-TAB2, anti-phospho-IKKα/β (Ser176/180), anti-phospho-MKK7 (Ser-271/Thr-275), and anti-MKK7 antibodies were purchased from Cell Signaling Technology. Anti-TAB3 antibody was from Abcam. Anti-Myc antibody, normal mouse IgG and normal rabbit IgG were from Millipore. Anti-Flag antibody (M2) was obtained from Sigma. Anti-HA, anti-α-Tubulin, and horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Rabbit anti-Vpr antibody was provided by National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. Mouse anti-p24 and anti-TAK1 were generated by immunizing mice with the corresponding full length proteins purified form E. coli BL21 (DE3). 4′, 6-diamidino-2-phenylindole (DAPI) and PMA were purchased from Sigma. Fluorescein-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. Serine/Threonine phosphatase inhibitor Calyculin A was purchased from Cell Signaling Technology.

Human immortalized nasopharyngeal epithelial cell NP460 and nasopharyngeal carcinoma cell lines, including CNE1, CNE2, C666-1, HNE1, HNE3, HK1, HNE2, HONE1, and SUNE1, were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai). The 293T cells for virus infection was purchased from American Type Culture Collection (ATCC, Manassas, VA). All cells were cultured by following the standard protocols. The primary antibodies including anti-TRAF2, p-MSK1, p-CREB, cyclin D1, CDK4, cyclin A, CDK2, cyclin B, CDK1, p27, p21, β-actin, Ki67, cleaved PARP, cleaved caspase-3, and secondary HRP-conjugated goat anti-mouse/rabbit IgG antibodies were products of Cell Signaling Technology (Danvers, MA). The SuperSignal™ West Dura Extended Duration Substrate was a product of Thermo Fisher Scientific (Waltham, MA).

Western blot analysis was performed according to our previously described protocol [21 (link)]. Primary antibodies included anti-TRAF2 (1:1000, Cell Signaling Technology 4724), anti-VEGFA (1:1000, Santa Cruz Biotechnology sc-7269), and anti-β-actin (1:2000, Proteintech 81115-1-RR).