4.11. Duolink In Situ Proximity Ligation Assay (PLA)

Duolink™ PLA (Sigma-Aldrich, St. Louis, MO, USA) was used for detecting the interaction between endogenous ABRACL and cofilin in situ within the cell. In this assay, two test proteins are first recognized by individual primary antibodies raised in different species. Next, two secondary antibodies conjugated with specific DNA-oligonucleotide (i.e., PLA probes) are added to bind to individual primary antibodies. If the two proteins of interest are close enough in situ, a connector oligonucleotide added into the reaction will hybridize and join the PLA probes to form a circular DNA structure. Following a ligation step, the circular DNA template can be amplified by DNA polymerase via a rolling-circle amplification process to generate copies of DNA for subsequent detection by fluorochrome-coupled probes [37]. For observing the interaction between ABRACL and cofilin in situ, cells were cultured on fibronectin-coated 18 mm chamber slides for 24 h before being fixed in 3.7% paraformaldehyde in PBS and permeabilized in 0.2% Triton X-100 in PBS. After incubation in the blocking solution for 1 h at 37 °C, washed in PBS, cells on the slides were incubated overnight with rabbit anti-ABRACL (1:400; Sigma-Aldrich Cat. #HPA030217, St. Louis, MO, USA) and mouse anti-cofilin primary antibodies (1:400; Santa Cruz E-8, Dallas, TX, USA) at 4 °C. After washing in TBST, PLA probes were added to the slides and PLA was performed following the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). The Duolink™ fluorescent detection red (Sigma-Aldrich, St. Louis, MO, USA) was used for the visualization of protein interaction. The slides were subsequently subjected to Hoechst staining for DNA or stained with FITC-phalloidin to visualize F-actin.