Ni nta beads

H1299 cells were transfected with plasmids encoding p53, HA-MDM2, His-Ub or Flag-SBDS as indicated in the figure legends and treated with MG132 for 4–6 hr before being harvested. At 48 h after transfection, cells were harvested and split into two aliquots, one for IB and the other for the ubiquitination assay. In brief, cell pellets were lysed in buffer I (8 M urea, 0.1 M Na₂HPO₄/NaH₂PO₄ (pH 8.0), 10 mM Tris-HCl (pH 8.0), 10 mM β-mercaptoethanol, 5 mM Imidazole) and incubated with Ni-NTA beads (Takara) that capture His-tagged proteins/complex at room temperature for 4 h. Beads were washed twice with buffer I, then twice with buffer II (8 M urea, 0.1 M Na₂HPO₄/NaH₂PO₄ (pH 6.3), 10 mM Tris-HCl (pH 6.3), 10 mM β-mercaptoethanol). The captured proteins were eluted and analyzed by IB with the indicated antibodies in the figure legends.

Lm harboring a 6His tagged Tim coexpressed with an untagged Mtz, under the regulation of a p_tetR promoter on the integrative pPL2 plasmid was grown at 37 °C in 50 mL BHI to an OD₆₀₀ of 0.5–0.6. The culture was then filtered using a 0.22 μm filter and the filtrate was incubated with 0.5 mL Ni-NTA beads (Takara, Kusatsu, Shiga, Japan) for 3 h at 4 °C with tilting. The Ni-NTA beads were then loaded on a column and washed with 10 mL of Buffer-P (0.3 M NaCl, 50 mM NaH₂PO₄, pH = 8) supplemented with 10 mM imidazole and then with 25 mM imidazole. The protein was eluted by 250 mM of imidazole in Buffer-P and precipitated by TCA. The proteins were separated on 12.5% SDS-polyacrylamide gels and stained with Coomassie brilliant blue to yield two primary visible bands: the full-length TIMP-like and its cleavage product. The stained band representing the cleavage product was isolated from the gel and analyzed by peptide mass fingerprinting at The Smoler Protein Research Center at the Technion, Haifa, Israel. Protein samples were digested by chymotrypsin, and the resulting proteolytic peptides were analyzed by LC–MS/MS on Q-Exactive (Thermo, Waltham, MA, USA) and identified by Discoverer software version 1.4.

Thrombin (T4648), all chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO), unless otherwise indicated. Transfection was done with Lipofectamine 2000 (Invitrogen). Ni-NTA beads were purchased from Clontech (635660). Anti-MAVS (Cell signaling, #3993), anti-IRF3 (Santa Cruz, sc-9082), anti-Flag (Sigma, F3165), anti-HA (Sigma, H9658), anti-GAPDH (Santa Cruz, sc-25778), anti-TBK1 (Santa Cruz, sc-52957; Cell signaling, #3504), anti-IKKε (Cell signaling, #2690S), anti-NEMO (Cell signaling, #2686S), anti-His (cw00c28), anti-IκBα (Cell signaling, #4814), anti-p-IRF3 (Epitomics, 2562–1), anti-p-TBK1 (Abcam, ab109272), anti-p-IκBα (Cell signaling, #2859), anti-p-IKKα/β (Cell signaling, #2078), anti-IKKα (Cell signaling, #2682), anti-IKKβ (Cell signaling, #2370), anti-NAP1 (Proteintech, 15042-1-AP), anti-TANK (Bioworld, BS2231), anti-SINTBAD (Cell signaling, #8605) antibodies were purchased as indicated. Antisera against viperion and p54/56 were generated by immunizing mice with the full length recombinant protein produced in E. coli, at Beijing Biotop Biotechnology, China.

Lac-mCherry was cloned into the pBAD-DEST49 expression vector (Clontech). A 6xHis C-terminal tag was added to aid in purification. BL21(DE3) cells were transformed and grown to mid-log at 37 °C with shaking in 2xYT media. At mid-log growth, expression of Lac-mCherry was induced with the addition of arabinose 0.1% (v/v) and the temperature was reduced to 15 °C and cells were allowed to grow overnight (approximately 12–16 h). Cell extract was purified with Ni-NTA beads (Clontech) and a sizing column (HiLoad 16/60 Superdex 75 Prep Grade with AKTA Prime FPLC) and purified Lac-mCherry was equilibrated into GF buffer (200 mM Tris pH 7.4, 200 mM KCl, 10 mM EDTA, 3 mM DTT).

293T cells were transfected with expression plasmids encoding FLAG-tagged p65, histidine-tagged ubiquitin and c-Myc-PDLIM1 or PDLIM2, and His-tagged proteins were purified as previously described33 . Briefly, transfected cells were extracted under denaturing condition with a buffer containing 6 M guanidium-HCl. Extracts were incubated with Ni-NTA beads (Clontech) for 2.5 hr and then washed with buffer containing 25 mM Tris pH 6.8, 20 mM imidazole. Purified proteins were subjected to immunoblot with anti-p65 or anti-c-Myc antibody.