Anti mouse minus

PLA was performed using the Duolink In Situ Green Kit purchased from Sigma-Aldrich (DUO92101) in accordance with the manufacturer’s instruction. Briefly, transfected cells were washed once with ice cold PBS, followed by fixation with 4% paraformaldehyde for 15 min at room temperature. Fixed cells were then washed three times with PBS and permeabilized with 0.3% Triton X-100 containing PBS for 10 min. Permeabilized cells were blocked with 5% normal goat serum for 1 hr at room temperature. The cells were then incubated with primary antibodies diluted in 10% normal goat serum supplemented with 0.1% Tween at 4°C overnight. Following the incubation, the cells were washed three times with PBS and then incubated with two PLA probes (Duolink In Situ PLA Probes Anti-rabbit PLUS and Anti-Mouse MINUS, Sigma-Aldrich) for 1 hr at 37°C. After probe incubation, the samples were incubated in ligation solution for 1 hr at 37°C. After ligation, cells were washed with Wash Buffer A and incubated in the amplification solution for 2 hr at 37°C. Cells were then serially washed twice in 1 × Wash Buffer B, 0.01 × Wash Buffer B once, and PBS once, followed by incubation with secondary antibodies for 1 hr at room temperature. Finally, cells were washed three times with PBS and mounted in Duolink In Situ Mounting Medium supplemented with DAPI. Fluorescence images were obtained with a confocal microscope.

The Duolink® PLA assay was performed as indicated in the manual. In brief, AGS cells were treated as indicated and stained with mouse anti-SQSTM1 and rabbit anti-LDHA antibodies as described for the immunofluorescent staining. Duolink® PLA was then performed using the anti-rabbit PLUS (#DUO92002, Sigma, St. Louis, MO, USA) and anti-mouse MINUS (#DUO92004, Sigma, St. Louis, MO, USA) probes. Following probe incubation, ligation, and amplification, the cells were observed and photographed under the confocal microscope (Olympus FV-1000).

Cells cultured on confocal dishes were fixed with 4% paraformaldehyde for 20 min at RT, permeabilized and incubated with primary antibodies at 4 °C. For in situ proximity ligation assay, protein–protein interactions between YAP or TAZ and p52 were detected with secondary proximity probes (Anti-Rabbit Plus and Anti-Mouse Minus) according to the Duolink in situ Fluorescence User Guide (Sigma-Aldrich).