Ab33915

The rainbow trout hepatoma cell line RTH-149 was obtained from the American Type Culture Collection (ATCC CRL-1710; Rockville, MD, USA) and cultured as previously described [33 (link)]. Escherichia coli 0111:B4 LPS and MG132 were purchased from InvivoGen (San Diego, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. The antibodies against JNK, phosphorylated JNK (Thr183/Tyr185), TAK1, phosphorylated TAK1 (Thr184/187), phosphorylated p38 MAPK (Thr180/Tyr182), K48-linkage specific polyubiquitin, K63-linkage specific polyubiquitin, HA-tag, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against p38 MAPK (ab170099) and TRAF6 (ab33915) were from Abcam (Cambridge, UK). The antibodies against 6×His tag and Flag-tag were from Invitrogen (Carlsbad, CA, USA).

Western blotting was performed using the standard SDS-PAGE separation technique as previously reported^{18 (link),19} . The harvested cells were disrupted with RIPA cleavage buffer (Cell Signaling Technology). Proteins were extracted from exosomes using a Total Exosome Protein Isolation Kit (Invitrogen, Carlsbad, CA, USA) following the instructions supplied. The lysates were collected after centrifugation, and the protein concentration was quantified with BCA kit (Thermo Fisher Scientific). 50 mg of protein was added to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with antibodies against phosphor-JNK (ab124956), phospho-MAPK (ab195049), phospho-ERK (ab201015), phospho-NF-κB (ab76302), TRAF-6 (ab33915) and GAPDH (ab8245) purchased from Abcam. Subsequently, the membranes were incubated with a secondary antibody for 1.5 h at room temperature. Immunoreactive protein bands were detected using an Odyssey scanning system (USA), quantified by Image J, and normalized to the corresponding amount of total protein. The experiment was repeated in triplicate with 3 replicates.

The experiments were performed according to the previous report [9 (link)]. Proteins were quantified using a BCA protein assay kit (Thermo Scientific, Waltham, MA), and 25 μg proteins were separated using 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). The antibodies for USP5 (1 : 500, #ab155993), TRAF6 (1 : 500, #ab33915), p65 (1 : 500, #ab16502), phos-p65 (1 : 500, #ab76302), IκBα (1 : 500, #ab32518), phos-IκBα (1 : 500, #ab133462), β-actin (1 : 1000, #ab179467), Myc (1: 1000, #ab32), Flag (1 : 1000, #ab49763), and K48-ub (1 : 500, #ab140601) were all bought from Abcam (Abcam, Cambridge, USA).

Whole-cell extracts were prepared in RIPA lysis buffer containing protease inhibitor cocktail (Roche), and lysis was reinforced using a syringe with a 0.33 × 13 mm needle. The total protein concentration was determined with DC Protein Assay (Bio-Rad Laboratories). An equivalent amount of proteins (750 μg/sample) was incubated with a rabbit primary antibody anti-TRAF6 (#ab33915, Abcam) for 3 h at 4°C. Streptavidin magnetic sepharose beads (#GE28, GE Healthcare Life Sciences) were incubated with a biotin-conjugated anti-rabbit IgG antibody (#ab64257, Abcam) for 2 h at 4°C. After washing, magnetic beads were mixed with the proteins, incubated with the primary antibody overnight at 4°C, and eluted. The purified protein concentration was determined with DC Protein Assay (Bio-Rad Laboratories) and followed with western blot assays (2 μg/well) to analyze TRAF6 interaction with RANK and TLR4.

MC-38 cells were cultured on coverslips (12 mm diameter) under normoxia (21% O₂) or hypoxia (0.2% O₂) for 8 h. Cells were washed with 1× PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Samples were washed twice with 1× PBS and permeabilized with 0.1% Tween-20 in 1× PBS for 10 min at room temperature. Duolink In Situ Orange Starter Kit Mouse/Rabbit (Sigma) was used together with a mouse-anti HIF1α (Clone: H1alpha67, Novus Biologicals) and rabbit-anti TRAF6 (ab33915, Abcam) antibodies were used at 20 µg/mL. Blocking and staining was performed following manufacturer's recommendations.