Far-western blot analysis

Purified bait protein TNT (2 μg) was separated using 12% SDS-PAGE and for further experiment electro-transferred onto the PVDF membrane. As described in protocol previously, briefly, the transferred bait protein TNT was denatured and renatured on the membrane. After blocking with 5% skimmed milk, the membrane with renatured bait protein was overlaid by prey protein IFT (3 μg ml⁻¹) in buffer (2% skimmed milk powder with 20 mM Tris (pH 7.6), 0.5 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.1% Tween-20,10% glycerol) for binding and incubated for 2 h at RT. Prey protein IFT was probed with primary mouse raised anti-IFT antibody (1:5000) followed by secondary antibody (dilution 1:5000, HRP-conjugated anti-mouse, Thermo Scientific, USA). Signal was detected by chemiluminescence using the Luminol reagent (Bio-Rad). Human protein ETHE1 (raised in-house; unpublished work) was used as negative control.