Protein-Protein Interaction Mapping via PLA

PLA was used to determine endogenous protein–protein interactions (118 (link)–120 ). As previously described in (121 (link)). Briefly, ChREBPα (by GenScript) and Pan-14-3-3 (Santa Cruz) antibodies were conjugated to Duolink oligonucleotides, PLUS and MINUS oligo arms, respectively, using Duolink^® In Situ Probemaker Following a PBS wash, cells were fixed with 4% formaldehyde solution for 10 min at room temperature, and blocked with Duolink Blocking Solution for 1 h at 37 °C and then incubated with 4 μg/mL ChREBPα-Plus and 14-3-3-MINUS overnight at 4 °C. PLA was performed according to the manufacturer’s directions. No secondary antibodies were used, because PLUS and MINUS oligo arms were directly conjugated to ChREBP and 14-3-3. Cells were imaged on a Zeiss 510 NLO/Meta system (Zeiss, Oberkochen, Germany), using a Plan-Apochromat 63×/1.40 oil differential interference contrast objective.