Duolink pla kit

Total cellular RNA was labeled for 3 h with EU (E10345; Thermo Fisher Scientific) or BrU (850187; MilliporeSigma). EU-RNA was detected with Alexa Fluor 488 azide or Alexa Fluor 594 azide using the Click-iT RNA Imaging Kit (C10329 and C10330; Thermo Fisher Scientific) according to the manufacturer’s instructions. When EU detection was combined with SAF-A IF, we performed the SAF-A IF first, followed by a 10-min fixation with 2% PFA in PBS. Coverslips were then processed for EU-RNA detection as described above. SAF-A:RNA complexes were detected in BrU-labeled cell populations using the rabbit α-SAF-A and mouse α-BrU antibodies in conjunction with the Duolink PLA kit (DUO92102; MilliporeSigma; Cy3-compatible fluorochrome).

From 25,000 to 50,000 cells were seeded on 35-mm glass-bottomed dishes (WPI). For substrate stiffness experiments, CytoSoft Imaging 24-well plates of 0.2 kPa and 64 kPa stiffness (Advanced Biomatrix) were used. The Duolink PLA Kit (Millipore Sigma) was used per manufacturer’s instructions. Specifically, the mouse minus, rabbit plus, and orange detection kit were used together. Cells were initially fixed with ice-cold 99% methanol. After the cells were treated per the PLA protocol, they were imaged within 24 h using an LSM 700 scanning confocal microscope (Carl Zeiss) with a 63×, 1.4 NA objective. Images were analyzed using Fiji through the “Analyze Particles” function or by measuring the raw integrated density of the entire cell, as indicated within the individual figures, and normalized to cell spread area as indicated in the figures.

To test whether epitopes of two proteins were located close to each other (<40 nm), the in situ proximity ligation assay (PLA) was performed using the Duolink PLA Kit (Millipore Sigma).²² (link)^,23 (link) Briefly, preadipocytes were cultured to preconfluence, fixed with cold methanol for 5 min, washed in PBS and incubated with the Duolink blocking solution for 30 min at 37 °C. Cells were further incubated for 30 min with a pair of antibodies (mouse anti-CD248 antibody (Proteintech, Chicago, IL, USA) and rabbit anti-insulin receptor β (IRβ) antibodies (E9L5V, Cell Signaling, MA, US)), each at 2 μg/ml. Controls included corresponding non-specific antibodies of the same species and isotype. After washes, the appropriate PLA probe Mix 1:40 was incubated for 1 h, followed by washes and addition of 25 μl of ligase for 30 min. Amplification was achieved by the addition of the DNA polymerase at 1:80 dilution for 100 min at 37 °C in the corresponding buffer. Slides were then mounted with DAPI for fluorescent imaging. Quantification was achieved by counting the number of fluorescent dots by ImageJ software set for a fixed area (magnification 60× objective), in a minimum of 7–10 random fields of interest per condition (n ≥ 3 experiments per condition). Fluorescence intensity was quantified with NIH ImageJ 1.50i imaging software (NIH, Bethesda, MD, USA).