Click it hpg alexa fluor 488 protein synthesis assay kit

Reagents were obtained from the following sources: antibodies to phospho-4E-BP1 (Thr37/46) from Cell Signalling (#2855); H3 antibodies from Abcam (#ab1791); anti-rabbit IgG secondary antibodies from KPL (#074–1506); Lambda Protein Phosphatase (Lambda PP) from New England Biolabs (#P0753S); ARTseqTM kit from Epicentre (#RPHMR12126); RNaqueous kit from Ambion (#AM1912); Torin 1 from Tocris (#4247); protease inhibitor cocktail from Sigma-Aldrich (P2714-1BTL); Halt™ Phosphatase Inhibitor Cocktail from Thermo Scientific (#78420); precast gels from BioRad (#4566033 and #4565013); To-Pro-3 iodide from Molecular Probes (#T3605); VECTASHIELD® from Vector Laboratories (#H1000); EdU from Thermo Fisher Catalog (#A10044); Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit from Thermo Fisher (#C10428); TMG-cap antibodies from Santa Cruz (#sc-32,724); Zymo RNA Clean & Concentrator-25 kit (#R1018); Ribo-Zero (#MRZG12324); Clarity Western ECL Substrate from BioRad (#170–5060).

A total of 1 × 10⁶ cells were seeded in 1 mL of medium in a 24-well plate for 24 h before additional treatment. Cells were stimulated for 6 h. After 6 h of stimulation, the medium was changed to l-methionine free Medium (Gibco DMEM supplemented with 200 µM l-cystine, 2 mM l-glutamine, 1 mM Sodium Pyruvate, 10 mM HEPES) with homopropargylglycine (HPG) according to the Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit (Thermo Scientific, Darmstadt, Germany) protocol. Cells were incubated for 30 min at 37 °C, 5% CO₂. Then, the cells were washed with DPBS and fixed with a 4% PFA fixation buffer (BioLegend, London, OK) for 10 min at RT. Cells were washed twice with 3% BSA in PBS and incubated with 200 µL permeabilization buffer (0.5% Saponin) for 10 min at RT. The washing step with DPBS was repeated twice. Cells were then incubated for 30 min protected from light in 250 µL Click-iT reaction cocktail containing Alexa Fluor 488 azide (recipe as prescribed on the kit protocol). The final washing step was carried out with the rinse buffer. The cells were scraped in 400 µL DPBS. FITC fluorescence levels were measured by FACS. Translation was inhibited by adding 10 µM cycloheximide (Santa Cruz Biotechnologies Inc., Dallas, TX, USA).

Nascent protein synthesis was detected using Click-iT^® HPG Alexa Fluor^® 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific). Root fragments (1.5 cm long) cut off from the control, 5-AU-treated, and 50 μM cycloheximide-treated onion seedlings were transferred to 50 μM water solution of l-homopropargylglycine (HPG; control) or to the mixture of HPG + 5-AU or HPG + cycloheximide, in dark. After 30 min, apical root parts were fixed in phosphate-buffered saline (PBS)-buffered 3.7% paraformaldehyde (4 °C; pH 7.4) for 45 min. For maceration, excised meristems were rinsed twice in PBS and transferred for 45 min to the citrate-buffered 2.5% pectinase. Next, root tips were squashed onto microscope slides (Polysine™, Menzel-Gläser) in a drop of distilled water, and placed on dry ice. After 15 min, coverslips were removed, and the slides were washed with PBS, distilled water, and air dried. Permeabilization of macerated cells was performed using 0.5% Triton X-100 for 15 min. HPG incorporation was detected using Click-iT^® reaction cocktail consisting of components prepared according to the vendor’s manual. Incubation was performed at room temperature for 30 min and, after that slides were washed in Click-iT® reaction rinse buffer and PBS. Cell nuclei were stained for 5 min with DAPI at a concentration of 0.4 mg mL⁻¹, washed in PBS and mounted in PBS/glycerol/DABCO mixture.