In situ detection reagents orange

PLA was performed with the Duolink (Sigma-Aldrich, USA) In Situ PLA technology–related kits, including In Situ Detection Reagents Orange (DUO92007), In Situ PLA Probe Anti-Rabbit MINUS (DUO92005), and In Situ PLA Probe Anti-Mouse PLUS (DUO92001). The exogenous interaction of cGAS and PRMT5 was detected in HEK293T cells cotransfected with HA-cGAS and Flag-PRMT5 plasmids, and the endogenous interaction of cGAS and PRMT5 was detected in macrophages derived from the freshly isolated mouse BMDMs. All fixation, incubation, ligation, and amplification procedures were done according to the manufacturer’s instructions.

All incubation and wash steps were performed using a BioShake iQ at 500 rpm. For washing, 50 μl of PBS were applied per well for 5 min. In vitro differentiated subcutaneous white adipocytes were permeabilized in TBS-T buffer (0.05% Tween) for 10 min and blocked with 2% BSA in PBS for 30 min and washed twice with PBS, respectively. Primary antibodies were applied for 1 h at RT in antibody diluent (PLA kit, Sigma, DUO92101). The following antibodies were used: anti-mouse insulin receptor (1:5, ThermoFisher CT-1), anti-rabbit integrin beta 1 (1:10, ThermoFisher, SA40-08), and anti-rabbit integrin beta 3 (1:40, ThermoFisher, SJ19-09). As a negative control, no primary antibody was used. The PLA assay was implemented as stated in the manufacturer's protocol, using the Duolink™ In Situ PLA® ProbeAnti-Rabbit PLUS, Anti-Mouse MINUS and the In Situ Detection Reagents Orange (Sigma, DUO92101). The staining solution had a final concentration of 25 μg/ml BSA, 1 μg/ml of DAPI (Thermo), and Lipitox 488 (HCS LipidTOXTM Green Neutral Lipid Stain (1:200, InvitrogenTM, H34775) in 1x saline-sodium citrate buffer. Cells were washed 3 times, and imaging was performed with a Leica confocal SP5. Quantification was performed by counting the dots per nuclei of adipocytes.

PLA on cultured cells was performed as described previously⁴³ using Duolink PLA probes and In situ Detection Reagents Orange (Sigma). Quantitative analysis was performed by taking pictures containing 40-70 cells using 40× objective lens of confocal microscope (SP5, Leica), and images of 6-8 representative fields from 3 independent experiments were collected. PLA particles were automatically counted by ImageJ (NIH) with the Analyze Particles mode.