AIPL1 Variant Binding Assay

Assays were performed as described in Sacristan-Reviriego et al.^{5 (link)}. Briefly, purified HSP90α or HSP90β (80 nM) were incubated in Immulon 4HBX 96-well plates (Fisher Scientific) for at least 1 h at 4 °C. 96-well plates were blocked using 1% blocking reagent (Sigma) in the same buffer (100 mM NaHCO₃ pH 8.5) for 1 h and then washed 3 times with chilled TBST (50 mM Tris HCl pH 7.5, 150 mM NaCl, 0.075% Tween-20) to remove excess protein. Cell lysates from transfected cells with wild type (w/t) AIPL1 or AIPL1 variants were prepared as described above and added to the wells in lysis buffer (20 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.075% Tween-20) containing 2% protease inhibitor cocktail. After a 1 h incubation at 4 °C, wells were washed 5 times with chilled lysis buffer. Incubation with mouse anti-myc antibody (1:1000) for 1 h was followed by 5 washes and another 1 h incubation with anti-mouse HRP conjugated antibody (1:10,000). After 5 final washes, the plate was incubated with substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma) for colorimetric detection at 30 °C for 30 min. The reaction was stopped using 0.5 M H₂SO₄ and the absorbance measured at 450 nm. For comparison of the AIPL1 variants to w/t AIPL1, the absorbance measured at 450 nm was normalised to the expression level in cell lysates detected by SDS-PAGE and western blotting.