Anti myc clone 9e10

Human embryonic kidney (HEK) 293T cells (ATCC) were co-transfected with NF-κB-luc reporter plasmid that contains an NF-κB response element upstream of the promoter driving the luciferase reporter gene, pGL4.74[hRluc/TK] control vector (Promega) and epitope tagged FLAG-HOIP or myc-HOIL-1L pcDNA3.1(+) plasmids in 6-well plates in triplicates using Lipofectamine 2000 transfection reagent. Since this assay could be carried out in a variety of cellular contexts, HEK293T cells were used because they are easy to transfect and suitable for the assay. The cells tested negative for mycoplasma contamination. Empty pcDNA3.1(+) vector was used as control. After 36 h, cells were lysed and 20 μl cell lysates were used to measure firefly luciferase and Renilla luciferase (transfection control) signals using the dual luciferase reporter assay system according to the manufacturer’s protocol (Promega). Data were analysed in GraphPad Prism and one-way ANOVA followed by Tukey post-tests were used for statistical analysis. Immunoblotting was performed with anti-FLAG (clone M2, Sigma-Aldrich) and anti-myc (clone 9E10, Sigma-Aldrich) antibodies, to confirm equivalent WT and mutant protein expression levels.

Cycloheximide-chase degradation assays were performed as described (97 (link)) with some modifications. Yeast cells were inoculated from an overnight culture at 0.05 OD₆₀₀/mL in 50 ml synthetic drop-out media, and were grown to mid-log phase (0.4–0.6 OD₆₀₀/ml). Cells corresponding to 20 OD₆₀₀ were pelleted at 2,000 g for 5 min and re-suspended to a final density of 2 OD₆₀₀/ml in 10 ml pre-warmed medium supplemented with 100μg/ml cycloheximide. A 2 ml aliquot was taken as the “0 min” time-point, pelleted, and flash-frozen in liquid nitrogen. The remaining culture was incubated in a shaker at 30°C and samples were taken at different time points and treated like the “0 min” sample. 250 ul of acid-washed glass beads (0.1mm, Bio-Spec) and 200 ul of lysis buffer (10 mM MOPS, pH 6.8, 1% SDS, 8 M urea, 10 mM EDTA, protease inhibitors) were added to the cell pellets. After vortexing for 2 min, 200 ul of urea sample buffer (125 mM Tris pH 6.8, 4% SDS, 8 M urea, 100 mM DTT, 10% glycerol, bromophenol blue) were added. The samples were incubated at 65°C for 5 min, centrifuged at 12,000 rpm, and subjected to SDS-PAGE and immunoblotting. HA- and MYC- tagged substrates were detected using anti-HA (clone 3F10, Roche) and anti-MYC (clone 9E10, Sigma) antibodies, respectively. PGK was used as a loading control by blotting with anti-PGK antibodies (Abcam).