Duolink kit

The co-localization of MTA1 with SMN, RPS3, YBX1 and PTBP1 was carried out using immunofluorescence detection. Briefly, the cells were grown on sterilized coverslips and were fixed by 4% paraformaldehyde at room temperature for 15 min, washed twice in PBS, permeabilized with 0.25% Triton X-100 at room temperature for 10 min and blocked with 0.5% bovine serum albumin for 30 min. The coverslips were subsequently incubated overnight with the primary mouse monoclonal MTA1 antibody and rabbit antibodies against SMN, RPS3 or YBX1 followed by a 1 h incubation in the corresponding fluorescent secondary antibodies diluted in blocking buffer. The coverslips were finally mounted with mounting medium containing DAPI. The images were acquired using fluorescence (Olympus) or confocal laser scanning (Leica) microscopy.
The in situ PLA experiments were performed to visualize the in situ interaction of MTA1 and SMN in the HCT116 cells using the Duolink kit (Olink Biosciences AB) and were performed according to the Duolink kit protocol. The images were acquired using fluorescence microscopy (Olympus).

In situ PLA experiments were performed to visualize the interaction of MTA1 and HDAC2 in cells using the Duolink kit (Olink Biosciences AB) as previously described[30 (link)]. The cover slips were incubated with primary antibodies (mouse anti-MTA1 and Rabbit anti-HDAC2) at room temperature for 1 h, washed 3 times for 5 min in PBS containing 0.1% Tween 20. Secondary proximity probes (Mouse- PLUS and Rabbit- MINUS) were incubated for 2 h at 37°C. The cells were washed 3 times for 5 min in PBS containing 0.1% Tween 20 at 37°C. All subsequent steps were performed according to the Duolink kit protocol (Olink Biosciences AB). The images were acquired using fluorescence microscopy (Olympus).

PLA was performed using the DUOLink^TM kit (OLINK; Uppsala, Sweden). H-Ras MCF-10A and MDA-MB-231 cells were seeded onto the glass slide and incubated for 24 h. After incubation, the cells were washed with PBS, fixed with 95% methanol/5% acetic acid for 10 min, permeabilized with 0.2% Triton X-100 for 5 min and again washed with PBS three times. Cells were then blocked with 5% BSA in PBS-T for 1 h and incubated with anti-STAT3 and anti-IKKα in PBS-T containing 1% BSA at 1:100 dilution at 4 °C overnight. After incubation, the cells were washed with PBS-T and then incubated with PLA plus and minus affinity probes for 1 h at 37 °C. The probes were then hybridized with a ligase. The DNA was amplified and detected under the fluorescence microscopy.