Eq0001

The cells were treated with puromycin (10 μg/ml, Invitrogen) for 10 min at 37 °C immediately before analysis. The samples were then scraped in ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold radioimmunoprecipitation (RIPA) buffer, after which the lysates were precleared by centrifugation at 15,000 × g for 20 min at 4 °C. The resulting lysates (20 μg) were subjected to SDS‒polyacrylamide gel electrophoresis followed by immunoblot analysis with an antibody against puromycin (Kerafast, EQ0001). The blot membrane was briefly stained with Fast Green FCF (Sigma) and imaged after protein transfer prior to blocking nonspecific sites with skim milk (5%, LPS solutions). All experiments were performed with at least three biological (independent) replications, and representative results are shown.

Before the addition of puromycin, immortalized MEF cells were globally activated with 452- nm band-pass filtered (#86-351, Edmund Optics) DIA lamp for 20 min. For puromycin labeling, cells were incubated with 3 µM puromycin (Tocris) in culture medium for 5 min at 37 °C, 5% CO₂ humidified microscope chamber. For the anisomycin control condition, cells were first incubated in 40 µM anisomycin for 20 min without blue light activation, and then puromycin labeling was conducted in the presence of 40 µM anisomycin and 3 µM puromycin for 5 min. After puromycin labeling, cells were quickly washed twice with warm 1X PBS-MC and then fixed with 4% PFA in 1X PBS-MC. After the fixation and washing, cells were permeabilized with 0.5% Triton X-100 in PBS-MC for 15 min followed by 2 times washing with PBS-MC. Cells were incubated with blocking buffer (4% goat serum in 1X PBS-MC) for 1 h at 37 °C. Cells were incubated with Anti-Puro (1:3000, EQ0001, Kerafast) and Anti-Beta-actin (1:1000, ab8227, Abcam) diluted in the blocking buffer, at 37 °C humidified chamber overnight. For the following rolling circle amplification, we used Duolink In Situ PLA Probe Anti-Rabbit Plus (DUO92002, Sigma-Aldrich), Anti-Mouse Minus (DUO92004, Sigma-Aldrich), and Detection Reagents Green (DUO92014, Sigma Aldrich) following manufacturer’s instruction as described in the previous studies^{49 (link),56 (link)}. Wash buffers 'A' and 'B' were equilibrated to room temperature before use. After incubation with antibodies, cells were washed two times with buffer 'A' for 5 min. After the vortexing of PLUS and MINUS PLA probes, each probe was diluted 1:5 in Duolink Antibody Diluent. PLA probe solution was applied to cells and cells were incubated in a pre-heated humidity chamber for 1 h at 37 °C. Following the incubation, cells were washed with buffer 'A' for 5 min twice. 5X Duolink Ligation buffer was diluted 1:5 in high-purity water. After the wash, Ligase was added to 1X Ligation buffer at a 1:40 dilution. Ligation solution was added to cells and cells were incubated in a pre-heated humidity chamber for 30 min at 37 °C. 5X Amplification buffer was diluted 1:5 in high-purity water. Following incubation, cells were washed with buffer 'A' for 5 min twice. After washing, Polymerase was added to 1X Amplification buffer at a 1:80 dilution. Amplification solution was added to cells and cells were incubated in a pre-heated humidity chamber for 100 min at 37 °C. After the incubation, cells were washed twice with buffer 'B' for 10 min. Extra wash with 0.01X buffer 'B' was performed for 1 min. Cells were imaged by microscope without mounting. For a more detailed description, please refer to Duolink PLA Fluorescence Protocol (Sigma-Aldrich).