Probe anti mouse plus

H1975 cells were seeded on 12-well plates containing 12-mm coverslips for 24 h before drug treatment. After 30 min or 24 h of drug treatment, the cells were washed twice with PBS Ca²⁺ Mg²⁺ and fixed with paraformaldehyde 4%, followed by PLA. The protocol of PLA was according to Chen and Huang [57 (link)]. Primary antibodies KEAP1 monoclonal antibody (1F10B6, Invitrogen) and NRF2 polyclonal antibody (ab31163, Abcam) were used at a dilution of 1:800. The secondary antibodies attaching probes were provided from the kit Duolink In Situ PLA Probe Anti-Rabbit MINUS (DUO92006, Sigma-Aldrich) and Probe Anti-Mouse PLUS (DUO92001, Sigma-Aldrich). The protein-protein interactions (PPIs) were detected with detection reagent FarRed (DUO92013, Sigma-Aldrich). Phalloidin-Alexa 488 nm and DAPI were used to visualize the cytoplasm and nucleus, respectively. Images were taken by using a spinning disk confocal microscopic system with a magnification of 60× objective. Total number of blobs was calculated as Z-projection via Metamorph software. A student’s t-test was applied to compare the difference between the control and each treatment group.

All antibodies used in this study were validated by conventional immunoblotting (see Figs S1 and S5). See Table S1 for antibody source and dilution used for detection in the NanoPro1000 with and without RCA/PLA and by immunoblotting. Recombinant human VEGFA (293-VE-010) was purchased from R&D Systems.
Horseradish peroxidase (HRP) conjugated anti-mouse and anti-rabbit antibodies were from ProteinSimple (#040-655, #040-656). The following secondary antibody-DNA conjugates were provided by Olink for Duolink In Situ PLA (the reagents are now available from Sigma Aldrich): Probe Anti-Mouse PLUS (Affinity Purified Donkey Anti-Mouse IgG, H+L; DUO92001), Duolink In Situ PLA Probe Anti-Rabbit PLUS (Affinity Purified Donkey Anti-Rabbit IgG, H+L; DUO92002), Duolink In Situ PLA Probe Anti-Goat PLUS (Affinity Purified Donkey Anti-Goat IgG, H+L; DUO92003), Duolink In Situ PLA Probe Anti-Mouse MINUS (Affinity Purified Donkey Anti-Mouse IgG, H+L; DUO92004), Duolink In Situ PLA Probe Anti-Rabbit MINUS (Affinity Purified Donkey anti-Rabbit IgG, H+L; DUO92005), Duolink In Situ PLA Probe Anti-Goat MINUS (Affinity Purified Donkey Anti-Goat IgG, H+L; DUO92006).