Duolink Proximity Ligation Assay for PKC-θ and ZEB1
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.
Dunn J., McCuaig R.D., Tan A.H., Tu W.J., Wu F., Wagstaff K.M., Zafar A., Ali S., Diwakar H., Dahlstrom J.E., Bean E.G., Forwood J.K., Tsimbalyuk S., Cross E.M., Hardy K., Bain A.L., Ahern E., Dolcetti R., Mazzieri R., Yip D., Eastgate M., Malik L., Milburn P., Jans D.A, & Rao S. (2022). Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers. Cancers, 14(6), 1596.
Corresponding Organization : QIMR Berghofer Medical Research Institute
Other organizations :
Australian Regenerative Medicine Institute, Monash University, St John of God Hospital, Canberra Hospital, Australian National University, Charles Sturt University, Monash Health, Peter MacCallum Cancer Centre, University of Melbourne, University of Queensland, Translational Research Institute, Royal Brisbane and Women's Hospital
Primary antibodies targeting PKC-θ-Thr538p and ZEB1
dependent variables
Protein-protein interactions between PKC-θ-Thr538p and ZEB1 as measured by the Duolink proximity ligation assay
control variables
Cells were fixed, permeabilized, and incubated with primary antibodies
Cells were processed according to the manufacturer's recommendations
Coverslips were mounted onto slides and examined as above
controls
Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned
Annotations
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