Click it protein synthesis assay kit

The protein synthesis assay was performed as described previously [33 (link)] using the Click-iT protein synthesis assay kit (C10428, Life Technologies) following the manufacturer’s instructions. Briefly, the MII-stage oocytes were incubated in culture medium supplemented with 50 μM HPG at 37° C with 5% CO2 for 1 h. Oocytes were fixed with 3.7% formaldehyde followed by permeabilization with 0.5% Triton X-100 for 20 min at room temperature. The HPG signal is indicative of the overall level of translation in oocytes.

Detection of nascent protein synthesis was assessed with Click-IT protein synthesis assay kit (#C10428, ThermoFisher) and performed following the manufacturer’s protocol. The methionine analog L-homopropargylglycine was added (50 µM) to cultures 30 min before the first sampling of each time point. The detection of click reaction was obtained by incubating fixed cells for 1 h with Alexa Fluor 488 azide in Click-iT cell reaction buffer solution. Fluorescence was recorded using a Leica TSC SP8 confocal microscope and ImageJ. Nascent protein synthesis was assessed by determining the signal intensity and finally normalized by the control condition of each biological replicate. Microscope settings (image exposure times and thresholds) were kept the same during the image acquisition of all the different conditions.

To observe translation in fungal cells, a Click-iT protein synthesis assay kit (ThermoFisher) was used per the manufacturer’s instructions. Details are provided in Text S1 in the supplemental material. Cells were imaged by DIC microscopy and by the use of the enhanced green fluorescent protein (EGFP) channel on a Zeiss Axio Imager.MI microscope (Carl Zeiss) at the same exposure time.

For nascent transcription assays, oocytes were incubated in M16 medium with IBMX supplemented with 1 mM EU (5-ethynyl uridine) for 2 h, fixed in methanol, then a Click-iT RNA Imaging kit (Thermo Fisher Scientific) was used and visualisation was by confocal microscopy. Changes in protein expression were monitored using a Click-iT Protein Synthesis Assay kit (Thermo Fisher Scientific). Oocytes and two-cell embryos were cultured in M16 medium with the methionine analogue HPG at a dilution of 1:500. Newly synthesised proteins incorporating the HPG were visualised by confocal microscopy.