Proximity Ligation Assay for LMTK2 and p35

Proximity ligation assays (PLAs) were performed with rabbit anti-LMTK2 and goat anti-KLC1, and with rabbit anti-p35 and goat anti-KLC1 antibodies using Duolink PLA probes and orange detection reagent (Sigma) according the manufacturer’s protocol. Briefly, cells were fixed and permeabilised as described above for immunofluorescence staining, blocked with PLA blocking buffer for 30 min at 37 °C and incubated with primary antibodies for 1 h. Following washing, samples were incubated with PLA probes for 1 h at 37 °C and after further washing, probes were ligated for 30 min at 37 °C and amplified for 100 min at 37 °C. Following PLAs, cells were incubated with tubulin primary antibody in PBS at 4 °C for 16 h, washed with PBS and incubated with fluorescent conjugated secondary antibody for 1 h in PBS. Following further washing, samples were mounted as described above. Control reactions to demonstrate the specificity of the assays involved omission of primary antibodies. Signals were quantified using ImageJ particle analysis function as described [42 (link)].