Any kdtm mini protean precast protein gels

Next, 100 µg of proteins from whole proteins extract, was incubated overnight with Phospho-Akt Substrate Sepharose^® Bead Conjugate (1:20; 9646; Cell Signaling; Danvers, MA, USA) to perform an immunoprecipitation. Beads were washed three times and were incubated at 100 °C for 5 min. The resultant immunocomplexes were resolved through precast SDS-PAGE (Any kDTM Mini-PROTEAN Precast Protein Gels, Bio-Rad); proteins were electrotransferred by a Transblot turbo system (Bio-Rad) for 30 min according to Bio-Rad protocol. Membrane was incubated with primary antibody: DJ-1 (1:1000; D29E5XP, Cell Signaling; Danvers, MA, USA). The detection of primary antibody was executed with antirabbit horseradish peroxidase conjugate secondary antibody (1:3000; Cell Signaling; Danvers; MA, USA). With SuperSignal West Femto ECL substrate (Pierce, Thermo Fisher Scientific Inc., Bremen, Germany); and images of blotted antibodies were acquired by Alliance 2.7 (UVITEC, Eppendorf, Milan, Italy). γ-Tubulin was used as loading control.

Nuclear DJ-1 interactors were pull down according with the immunoprecipitation proteins previously reported. DJ-1 interactors were resolved by SDS-PAGE (Any kDTM Mini-PROTEAN Precast Protein Gels, Bio-Rad) and visualized using EZBlue Gel Staining Reagent (Sigma Aldrich). Gel lane referred to DJ-1-IP and Mock-IP were sliced according to molecular weight. Gel bands were processed according to the protocol reported in the Section 2.6.
Proteins identified in the Mock-IP gel line were classified as unspecific interactors and eliminated from the list of specific DJ-1-nuclear interactors.