Pla probe anti rabbit minus

HeLa cells were cultured on coverslips then washed with cold PBS and fixed with cold methanol for 20 min at −20 °C. Coverslips were washed again 4 times with cold PBS. All incubations were performed in a humidity chamber. Blocking was done with 0.1% tween-TBS with 1% BSA for 1 h at 37 °C. Incubations with primary antibodies were performed overnight at 4 °C. Cells were washed three times for 3 min with 0.1% tween-TBS with gentle agitation. Incubations with PLA Probe Anti-Mouse PLUS (Sigma-Aldrich, #DUO92001) and PLA Probe Anti-Rabbit MINUS (Sigma-Aldrich, #DUO92005) were performed for 2 h at 37 °C. Next, coverslips were washed three times for 3 min in T-PBS buffer under gentle shaking and then incubated with DNA ligase previously diluted in ligation buffer for 30 min at 37 °C according to manufacturer’s instructions. Coverslips were washed three times for 3 min in T-PBS buffer under gentle shaking and incubated with a DNA polymerase previously diluted in amplification buffer for 90 min at 37 °C. Finally, coverslips were rinsed for 10 min in the presence of DAPI under gentle shaking and then washed 2 min with 2X and then 0.02X SCC buffer (30 mM sodium citrate, 300 mM sodium chloride). Coverslips were mounted in Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, #F4680) and analyzed using a Zeiss LSM confocal microscope.

The Duolink proximity ligation assay was employed using PLA probe anti-mouse PLUS (DUO92001), PLA probe anti-rabbit MINUS (DUO92005), and Duolink In Situ Detection Reagent Red Kit (DUO92008) (Sigma Aldrich). Cells were fixed, permeabilized, and incubated with primary antibodies targeting LSD1 and ACE2. Cells were processed according to the manufacturer’s recommendations. Finally, coverslips were mounted onto slides and examined as above.

Using our previously established protocols [34 (link),35 (link)], the Duolink proximity ligation assay was employed using PLA probe anti-mouse PLUS (DUO92001), PLA probe anti-rabbit MINUS (DUO92005), and Duolink In Situ Detection Reagent Red Kit (DUO92008) (Sigma-Aldrich). Cells were fixed, permeabilized, and incubated with primary antibodies targeting PKC-θ-Thr538p and ZEB1. Cells were processed according to the manufacturer’s recommendations. Finally, coverslips were mounted onto slides, which were examined as above.