![]() The principle of the technology is based on two unique probes provided with the Duolink kit. The technique was also applied on other cell lines (HEK293T and Cos7) with similar results (data not shown). We concluded that the number of spots provide an accurate comparative measurement of signaling levels over time. We counted the signals (fig 1F) and compared the measurement with the quantification obtained from immunoblot of the same cells 8 (figure 1G). This method allowed us, for the first time, to see the activation of BMP effector-complexes over time and not just the presence of the phosphorylated Smad1/5/8, which may not all be engaged in active complexes with Smad4. We used different tissue culture cells including Neuro2a and relied on commercial antibodies raised in different species (mouse a-Smad4 and rabbit a-P-Smad1/5/8) to achieve this (fig 1). Here, we demonstrate how to use PLA not only to visualize but also to quantify endogenous complexes between Smad effectors activated downstream of BMP stimulation over time. ![]() PLA is a new technique that researchers have started using in different systems with great success 6, 7. Co-localization of antibodies exhibits low resolution and cannot be used to visualize true interaction. It has not been possible to visualize and quantify complexes between two endogenous proteins in situ with immunofluorescence before. ![]() The visualization of protein complexes in situ is in great demand, particularly for studies in signaling where protein interaction and protein modification are the means that cells use for sending a signal from their surface to the nucleus. Note that the a-P-Smad1/5/8 antibody cannot distinguish between the 3 different proteins present in complex with Smad4. The cells were lysed and the proteins were subjected to SDS-PAGE and analysed by immunoblotting with a-P-Smad1/5/8 and a-PCNA as loading control. (G) Neuro2a cells were left untreated or treated with Dorsomorphin (2 μM) or BMP4 (25 ng/ml) for 60 min. (F) The PLA signals were counted with the Duolink ImageTool software and the average number of spots in the nucleus per cell is presented in the graph. Pictures were obtained with a Leica SP5 confocal microscope. The primary antibodies a-P-Smad1/5/8 alone (C) and a-Smad4 alone (D) or omission of the primary antibodies (E) were used as controls. Antibodies against P-Smad1/5/8 and Smad4 were used to detect the active complexes in A and B. Cells were treated with 2 μM dorsomorphin (inhibitor of the BMP pathway) (A) or 25 ng/ml BMP4 (B) for 60 min or left untreated (C-E). In situ PLA on Neuro2a cells after BMP4 stimulation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.įigure 1. in a complex, which occurs only with signaling activation. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. The number of signals can be counted and compared providing a measurement. ![]() It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. ![]() In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. BMPs are responsible for a wide range of developmental and biological effects. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |