After 24 h replace the medium with serum free GMEM (supplemented with 0.1% albumin, 1x NEAM, 1x sodium pyruvate and 1x L-glutamine) for Neuro2a or with serum free DMEM (supplemented with L-Glutamine and 1x Penicillin/Streptomycin) for HEK293T/Cos7 and incubate the cells for 2-3 h.Incubate the cells at 37☌ in a humidified, 5% CO 2 incubator.Split the cells using trypsin (0.05%-EDTA) and plate 20.000-25.000 cells per well in 50 μl medium. Sterilize polysine slides by immersing them in 70% ethanol and using Immedge pen draw 1 cm 2 wells. Split the cells using trypsin (0.05%-EDTA) and plate 15.000-20.000 cells per well in a 16-well chamber slide.Ĭulture HEK293T or Cos7 cells in DMEM supplemented with 10% FBS, 1x L-glutamine and 1x Penicillin/Streptomycin. Culture Neuro2a cells in GMEM supplemented with 10% FBS, 1x Non-Essential amino acids (NEAM), 1x sodium pyruvate and 1x L-glutamine.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.ġ. 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.
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