RT reactions were performed using The SuperScript III First-Strand Synthesis System (Life Technologies) and total RNA from TH9 cells at 24 h of differentiation. PaltinumTaq DNA polymerase high fidelity (Invitrogen) was used for PCR amplification of desired fragments corresponding to full-length p62 (p62_FL) and p62 lacking the UBA domain (p62_UBA∆). All forward primers had a T7 promoter sequence: 5′-taatacgactcactatagggaga-3′ while reverse primer had a TAA stop codon. The primers were:
p62_FL: forward 5′-ataaaagctgggctctcggc-3′; reverse: 3′-tcacaatggtggagggtgctt-5′ and p62_UBA∆: forward 5′-atggcgtcgttcacggtgaa-3′; reverse: 3′-cttcagccctgtgggtcctt-5′.
Synthetic p62_FL and p62_UBA∆ proteins were then generated using TnT Quick Coupled Transcription/Translation Systems (Promega L1170) combined with biotinylated lysine (Transcend tRNA Promega L5061) according to the manufacturer’s instructions. These two proteins were subsequently incubated with total extracts from TH9 cells differentiated for 24 h for pull-down assay. For this we used the Pierce Pull-down Biotinylated-protein: protein interaction kit (Thermo Scientific 21115) according to the manufacturer’s instructions. The binding of endogenous PU.1 and LC3-II was revealed by western blots.
p62_FL: forward 5′-ataaaagctgggctctcggc-3′; reverse: 3′-tcacaatggtggagggtgctt-5′ and p62_UBA∆: forward 5′-atggcgtcgttcacggtgaa-3′; reverse: 3′-cttcagccctgtgggtcctt-5′.
Synthetic p62_FL and p62_UBA∆ proteins were then generated using TnT Quick Coupled Transcription/Translation Systems (Promega L1170) combined with biotinylated lysine (Transcend tRNA Promega L5061) according to the manufacturer’s instructions. These two proteins were subsequently incubated with total extracts from TH9 cells differentiated for 24 h for pull-down assay. For this we used the Pierce Pull-down Biotinylated-protein: protein interaction kit (Thermo Scientific 21115) according to the manufacturer’s instructions. The binding of endogenous PU.1 and LC3-II was revealed by western blots.