Phenanthroline

PNI day 7 iNeurons (without glia) were lysed in TUBE lysis buffer [5 nM Tris-HCl, 0.15 M NaCal, 1 mM EDTA, 1% NP-40, 10% glycerol, cOmplete mini protease inhibitor cocktail (Roche), 50 µM pr-619 (LifeSensors), 1×1,10-phenanthroline (LifeSensors)]. 850 µg protein was incubated with 80 µL equilibrated TUBE1 magnetic beads (LifeSensors) at 4 °C for 2 h. Beads were then washed 3X in TBST, resuspended in 1× Lamelli sample buffer with ß-mercaptoethanol and boiled for 8 min at 95 °C. Samples were centrifuged and transferred to a new tube and used for western blotting.

Cells were stimulated with either plate bound 5 μg/ml anti-CD3 and soluble 2 μg/ml anti-CD28 or by anti-CD3/CD28 dynabeads (1 bead:3 cell) in presence of 10 ng/ml IL-4 (Peprotech) or 20 μg/ml anti-IL-4 or 10 ng/ml IL-4 along with 1 μM NAEi (Lifesensors; S19830) for 4 h. Cells were then lysed in NP-40 buffer (50 mM Tris-cl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 1% NP-40) in the presence of deubiquitylase inhibitor PR619 (Lifesensors; S19619), 1,10 phenanthroline (Lifesensors; S19649), Protease inhibitor cocktail EDTA free (Roche) and phosphatase inhibitor cocktail (Thermo). After 30 min of lysis, the samples were centrifuged at 15,000 rpm for 10 min at 4 °C. Cleared supernatant was quantified using Bradford reagent and 3–4 mg protein was used for Immunoprecipitation. For preclearing, supernatants were incubated with 2 μg anti-IgG conjugated with protein A dynabeads for 2 h at room temperature. Precleared supernatant was then incubated with either anti-Cul5 (2 μg), or anti-CIS (3 μg) or anti-IgG (2 or 3 μg) conjugated with protein A dynabeads for overnight at 4 °C. Beads were then washed five times with 1X TBS containing 0.1% Tween 20. The immunoprecipitated proteins were eluted with 2X Laemmli buffer (240 mM Tris, pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% β-mercaptoethanol, 40% glycerol) and boiled at 95 °C for 5 min.