Tnt quick coupled system

For the production of recombinant proteins, mouse p62 and human Cep63 were cloned into a pBSK–Flag(3 × ) vector. Flag–Cep63 and p62 were in vitro translated using TNT Quick Coupled System (Promega). For the immunoprecipitation assay, cell extracts were prepared from MEFs transfected with Flag–Cep63 or GFP–Cep63. Lysates or in vitro-translated proteins were then incubated with anti-p62 antibody and normal rabbit IgG as a negative control for 2 h at 4 °C. Immune complexes were captured with Dynabeads protein G (Invitrogen) and were washed four times. The co-immunoprecipitation of Cep63 was detected by Western blotting using anti-Flag or anti-GFP antibody.
For the close proximity assay, a Duolink proximity ligation assay was performed according to the manufacturer's instructions. Briefly, Flag(3 × )–Cep63-expressing cells were grown on coverslips and were fixed in 99% cold methanol for 15 min. The cells were then permeabilized in PBS containing 0.5% NP-40 for 15 min. After 30 min blocking in PBS with 0.2% coldwater fish gelatin and 0.5% BSA, primary antibodies were applied. After washing the cells, proximity ligation assay probes were added, which was followed by hybridization, ligation and amplification at 37 °C. Fluorescence images were then acquired and were quantitatively analysed with the In Cell Analyser 2000 (GE Healthcare).