Azide fluor 488

Antibodies and sources: CDK9, pCDK9, RNAPII, phospho-RNAPII, TGM2, Cyclin B1, Cyclin E1, and NUP98 (Cell Signaling Technology); CDC37, SPT5, Stomatin, PLK1, Cyclin A1 (Santa Cruz Biotechnology); BRD4, Cyclin T1 and GFP (Abcam); Caspase-8 (Enzo Life Sciences); β-Actin, pCDK9, Vimentin, and Flag (Sigma-Aldrich).
Reagents and sources: CellTiter-Blue Cell Viability assay and Caspase-Glo 3/7 assay (Promega); BrdU kit (Roche); Thymidine, 5-ethynyl uridine (EU), Azide-fluor 488 (Sigma-Aldrich); AnnexinV and 7AAD (BD); [γ-32P] ATP (3000Ci/mmol, Amersham Pharmacia); Trail, FasL (Enzo Life Sciences); PLA assay kit (Olink Biosciences); BAY1251152, Cisplatin, and Carboplatin (Selleckchem); BioCoat Matrigel invasion chamber (Corning); Migration chamber (Ibidi); RNeasy Plus kit (Qiagen), active GST-CDK9/Cyclin K (SRP5012, Sigma-Aldrich).
The following vectors were used: pCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene); p3xFlag-CMV-7.1 (E7533, Sigma); pGEX-5X-3 (28–9545-55, GE Healthcare) and pEGFP-C2 (6083-1, Clontech). All siRNAs and primers were from Sigma-Aldrich.

DMSO (0.5 μL) was added to rat intestinal mucosal protein fractions or intestinal lipid rafts from Caco-2 cells at a rate of 1 mg/mL, followed by incubation on ice for 15 min. Subsequently, 10 mM IIXEK (0.5 μL) dissolved in DMSO was added, again followed by incubation on ice for 15 min. Photoaffinity labeling was performed as previously described [18 (link)]. Then, the protein fractions and lipid rafts were subjected to UV irradiation at 365 nm for 30 min, and 302 nm for 10 min. Later, 2.5 μL of 10% SDS and 0.5 μL of 5 mM Azide-fluor488 (Sigma-Aldrich) were added. Next, 2 μL of the catalytic mixture [1.5 μL of 1.7 mM TBAT (TCI), 0.5 μL of 50 mM CuSO4 (Sigma-Aldrich), and 0.5 μL of 50 mM Sodium l-ascorbate (Sigma-Aldrich)] were added, followed by incubation at 32 °C for 30 min. Subsequently, this mixture was subjected to 4–12% SDS-PAGE for fluorescence scanning. The BenchMark™ Fluorescent Protein Standard was used as the molecular weight marker. The photoaffinity labeled protein was analyzed by nano LC-MS/MS (Q Exactive Plus, Thermo Scientific, Waltham, MA, USA). A Mascot search engine (www.matrixscience.com) was used for the database search within UniprotKB/Swiss-Prot database to match with the parent proteins.

Cells were incubated in the presence of inhibitors for the corresponding amount of time. Ethylene-deoxyuridine (10 µM; ThermoFisher) was added to the media 1 h before fixation. Subsequently, the cells were trypsinized and fixed in ice-cold absolute ethanol. The cells were rehydrated via a PBS wash and permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature with rotation. After a PBS wash, a click chemistry reaction cocktail was added to the cells (100 mM Tris–HCl pH 7.6, 4 mM CuSO₄, 2.5 µM azide–Fluor 488 (Sigma), 100 mM sodium ascorbate (Sigma)) and incubated for 30 min at room temperature, protected from light. After a PBS wash, propidium iodide/RNase staining solution (Thermo) was added to the cells for 30 min. The cell-cycle profiles were acquired on a BD LSRII flow cytometer and analysed using the BD FACSDiva software.

3^rd instar larvae were flipped and incubated in Schneider’s insect medium [Sigma, S0146] containing 10μM 5-Ethynyl-2’-deoxyuridine (5-EdU) [Jena Bioscience, CLK-N001] for 30 minutes. The larvae were then fixed and permeabilized as described earlier. Larvae were then incubated with 250μl click reaction cocktail (29μl 100mM ammonium guanidine, 10μl 100mM copper sulfate, 10μl 50mM THPTA [Sigma, 762342], 25μl freshly made 1M sodium ascorbate and 1μl 6.2mM Azide-fluor 488 [Sigma, 760765] or Cy5-Azide [Sigma, 777323] in 175μl HEPES buffer) for 30 minutes. The larvae were then processed for immunofluorescence or directly counter-stained with DAPI. Eye imaginal discs were then dissected and mounted in Vectashield.