Irdye800cw azide infrared dye

Cells were pretreated with 200 µM competitor for 15 minutes (or 3 h for 5 and 6) where applicable and then treated with 20 µM PRP or DMSO control. After incubation with the PRP at 37 °C for 30 min (or 3 h for PRPs 13 and 14), the cells were cooled on ice and irradiated with 366 nm light for 30 min. The medium was removed, and the cells gently washed twice with 2 mL PBS and then covered with 1 mL PBS. Cells were scraped/transferred from the plate into Eppendorf tubes, spun down at 1,000 g for 5 minutes at 4 °C, the supernatant removed, and the cells resuspended in whole cell lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 5% glycerol and 1x protease inhibitor cocktail (Roche)) and 1x phosphatase inhibitor cocktail (ThermoFisher)). Samples were homogenized by vortexing, incubated on a rotating stand at 4 °C for 1 h, then spun down at 19,000g for 10 minutes at 4 °C and the protein concentrations were determined by bicinchoninic acid (BCA) assay. Next, 35 µg total lysates were incubated with IRDye® 800CW Azide Infrared dye (LI-COR) at concentration equal to that of the PRP, TCEP (1 mM), TBTA (0.1 mM), and CuSO₄ (1 mM) for 90 minutes at room temperature. The samples were then frozen at −20 °C until analyzed by Western blot.

Cells were lysed with 2% SDS, extracts were quantified and diluted to 1% SDS, and 100 μg of protein was subjected to click reaction using Click-it Kit (Thermo Fisher) and IRDye 800CW Azide Infrared Dye (Li-COR). Samples were incubated in dark at room temperature for 1.5 h, and subsequently, the proteins were precipitated using standard methanol/chloroform protocol. Pellets were air-dried and re-suspended in 1% SDS. 20 μg of protein were boiled with sample buffer and loaded onto SDS-PAGE gel for further analysis of inputs or were incubated with antibody against KRT20 (Atlas antibodies, HPA024309) overnight at 4°C with shaking. The next day, protein A beads were added, and samples were incubated 1 h under same conditions. Subsequently, after extensive washes, samples were boiled with sample buffer and loaded onto SDS-PAGE gel followed by standard WB protocol. In order to detect KRT20, membranes were incubated for 1 h in dark at room temperature with 1:5000 Goat anti-Rabbit Alexa Fluor 680 (Thermo Fisher). Membranes were washed 3 times with TBS-T 0.2% for 10 min and visualized using Odyssey Fc Imaging system (Li-COR).