Duolink in situ pla probe anti rabbit minus

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.

All antibodies used in this study were validated by conventional immunoblotting (see Figs S1 and S5). See Table S1 for antibody source and dilution used for detection in the NanoPro1000 with and without RCA/PLA and by immunoblotting. Recombinant human VEGFA (293-VE-010) was purchased from R&D Systems.
Horseradish peroxidase (HRP) conjugated anti-mouse and anti-rabbit antibodies were from ProteinSimple (#040-655, #040-656). The following secondary antibody-DNA conjugates were provided by Olink for Duolink In Situ PLA (the reagents are now available from Sigma Aldrich): Probe Anti-Mouse PLUS (Affinity Purified Donkey Anti-Mouse IgG, H+L; DUO92001), Duolink In Situ PLA Probe Anti-Rabbit PLUS (Affinity Purified Donkey Anti-Rabbit IgG, H+L; DUO92002), Duolink In Situ PLA Probe Anti-Goat PLUS (Affinity Purified Donkey Anti-Goat IgG, H+L; DUO92003), Duolink In Situ PLA Probe Anti-Mouse MINUS (Affinity Purified Donkey Anti-Mouse IgG, H+L; DUO92004), Duolink In Situ PLA Probe Anti-Rabbit MINUS (Affinity Purified Donkey anti-Rabbit IgG, H+L; DUO92005), Duolink In Situ PLA Probe Anti-Goat MINUS (Affinity Purified Donkey Anti-Goat IgG, H+L; DUO92006).

To examine SL-13R-protein interaction, proximity link assay was performed. UCB CD34+ cells were cultured with biotin-conjugated SL-13R for 48 h. Cytospin, cell fixation and permeabilization are similar to previous mention in Immunocytochemistry. After overnight incubation with rabbit anti-AHNAK, ANXA2, and PLEC antibody (Table S4) and mouse anti-biotin antibody, Duolink In Situ PLA Probe anti-rabbit MINUS and anti-mouse PLUS were applied to samples (Sigma-Aldrich, Uppsala, Sweden). Proximally located antibody-binding probes were ligated with oligonucleotide by ligase at 37℃ for 30 min using Duolink In Situ Detection Reagent Green (Sigma-Aldrich, Uppsala, Sweden). Rolling Circle Amplification of probes was performed using DNA polymerase at 37℃ for 100 min. After wash with Buffer B, nuclei were stained with TOTO-3, and analysed using a FluoView 1000 Confocal Microscope.

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)}.

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.