Duolink in situ pla probe anti mouse plus

The PLA was performed according to the manufacturer’s manual and the following reagents were used: Duolink^® In Situ Detection Reagents Red (Sigma-Aldrich, Darmstadt, Germany; #DUO92008-100RXN), Duolink^® In Situ PLA^® Probe Anti-Mouse PLUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92001-100RXN) and Duolink^® In Situ PLA^® Probe Anti-Rabbit MINUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92005-100RXN). In short, the cells were co-stained over night at 4 °C using the following antibodies: SETD1B (Abcam, Cambridge, UK; #ab113984; 1:1000) and ZEB1 (R&D Systems, Wiesbaden, Germany; #639914; 10 µg/ml). The next day, the PLA probe solution was added to the cells as described in the protocol. After the ligation and amplification steps, the nuclei were stained using ProLong® Gold Antifade reagent (Life Technologies, Darmstadt, Germany). Detection was performed using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokyo, Japan).

Detection of an interaction between BRCA1 and the cavin or CAV1 proteins was assessed using the Duolink II Detection Kit (Sigma-Aldrich) according to the manufacturer's specifications. The Duolink In situ PLA Probe Anti-Rabbit MINUS (Sigma-Aldrich, DUO92005, RRID:AB_2810942) and Duolink In situ PLA Probe anti-Mouse PLUS (Sigma-Aldrich, DUO92001, RRID:AB_281039) and Duolink In situ detection reagents Orange (DUO 92007) were used in all PLA experiments. The primary antibodies used were mouse monoclonal GFP (1:500) and rabbit polyclonal BRCA1 (1:200), rabbit cavin3 (1:200) and mouse PCNA (1:100), rabbit cavin3 (1:200) and mouse Aurora Kinase (1:100), rabbit cavin3 (1:200) and Flotillin (1:100), and cavin3 (1:200) and mouse cavin 1 (1:100). The signal was visualized as a distinct fluorescent spot and was captured on an Olympus BX-51 upright Fluorescence Microscope. The number of PLA signals in a cell was quantified in ImageJ using a Maximum Entropy Threshold and Particle Analysis where 50 cells in each treatment group were analyzed from at least three independent experiments.

Duolink PLA in situ fluorescence (Sigma-Aldrich) was performed according to the manufacturer’s instructions with Duolink in situ PLA probe anti-mouse PLUS (#DUO92001), Duolink in situ PLA probe anti-rabbit MINUS (#DUO92005), Duolink in situ detection reagents Red (#DUO92008) and Duolink in situ wash buffers-fluorescence (#DUO82049). Pulmonary fibroblasts from Bmi-1^−/− mice were treated with TGF-β1 and detected with antibodies against p16 (MA5-17142, Thermo Fisher Scientific, IL, USA) and ERK1/2 (#4695, Cell Signaling Technology), and p16 (MA5-17142, Thermo Fisher Scientific, IL, USA) and pERK1/2(Thr202/Tyr204) (#4370, Cell Signaling Technology). The PLA signal (λex 594 nm, λem 624 nm; Texas Red) was analyzed as previously described^{28 (link)}.

To visualize the subcellular localization of the ILK–RhoT1 interaction, MDA-MB-231 cells were stably transfected with pPB vectors encoding mEmerald-ILK and V5-RhoT1. To avoid mislocalization of ILK by its excessive expression, mEmerald-ILK cDNA was placed downstream of the TRE3G promoter, and expression was induced by the addition of 10 nM doxycycline 1 h after plating. After 48 h, cells were sequentially fixed with 2% PFA and −20 °C methanol, and blocked with MAXblock as described above. After washing in PBS, cells were incubated with an anti-ILK mouse mAb (1:50) and anti-V5 rabbit mAb (1:50) in 5% BSA/PBS for 16 h at 4 °C. PLA reactions were performed using Duolink® In Situ PLA Probe Anti-Mouse PLUS, Anti-Rabbit MINUS, and Detection Reagents Orange (Sigma), following the manufacturer’s instructions. After the ligation and amplification reactions, cells were further incubated with the anti-TOMM20 antibody conjugated with Alexa 405 (1:100) and DRAQ5 (1:5000) in Duolink Antibody Diluent (supplied in the kit), to visualize mitochondria and nuclei, respectively. Specificity of the antibodies used for PLA was confirmed by immunostaining performed at the same concentrations.

The proximity ligation assay was used to visualize AhR/SRC complexes in SK28 cells. The cells, grown on glass coverslips, were fixed with 4% PFA in 0.1 M phosphate buffer (15735‐60S, Electron Microscopy Sciences) for 15 min at RT and PLA performed using the Duolink® in Situ detection Reagent Orange (DUO92007), Duolink® in Situ PLA® Probe Anti‐Mouse PLUS (DUO92001), and Duolink® in Situ PLA® Probe Anti‐Rabbit MINUS (DUO92005), SIGMA kits according to the manufacturer's protocol. After blocking, the reaction was performed with the primary antibodies: mouse anti‐AhR (C20, 1/100) and rabbit anti‐SRC (1C12, 1/100). Following the ligation and amplification steps, the coverslips were immobilized on microscopic slides using mounting medium containing DAPI. The ligation step was omitted in the control. Imaging analysis was carried out using a delta vision system (Applied Precision). The number of foci was quantified for at least 30 cells.

The proximity ligation assay was applied in order to visualize AhR/ARNT complexes in MCF7 cells. The cells, grown on glass coverslips, were fixed with 4% PFA in 0.1 M phosphate buffer (15735-60S, Electron Microscopy Sciences) for 15 min at RT and PLA was performed using the kit ((DUO92007) Duolink® in Situ Detection Reagent Orange, (DUO92001) Duolink® in Situ PLA® Probe Anti-Mouse PLUS, (DUO92005) Duolink® in Situ PLA® Probe Anti-Rabbit MINUS, SIGMA) according to the manufacturer’s protocol. After blocking, the reaction with primary antibodies, mouse anti-AhR (C20, 1/100) and rabbit anti-ARNT (1C12, 1/100). Following the ligation and amplification steps, the coverslips were immobilized on the microscopic slides with the mounting medium containing DAPI. In control experiment, the ligation step was omitted. Imaging analysis was carried out using a delta vision system (Applied Precision). Number of foci was quantified in at least 30 cells.