Rabbit plus and mouse minus duolink in situ pla kits

Proximity ligation was performed using the Rabbit PLUS and Mouse MINUS Duolink In Situ PLA kits (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, KYSE180 cells (5 × 10⁴) were plated on coverslips and treated with nicotine or vehicle for 24 h. The cells were then washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature (RT). Subsequently, cells were washed with TBS (25 mM Tris, 100 mM NaCl, pH 7.4), incubated for 10 min in 50 mM NH4Cl, TBS, washed with TBS, permeabilized for 15 min in 0.1% Triton X-100 in TBS, washed with TBST (0.05% Tween 20 in TBS), and then blocked for 2 h with 0.5% milk powder in TBST in a humidified chamber at RT and incubated overnight at 4 °C with anti-OTUD3 (Sigma-Aldrich, HPA028544) and anti-ZFP36 (Abcam, ab124024) antibodies. After washing with TBST, proximity ligation was performed using the PLA kits (Sigma-Aldrich). Cells were further counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich) to visualize the nuclei. The images were obtained by using laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging, Oberkochen, Germany).

The cells were grown on collagen-coated Lab-Tek II chamber slides (Nunc, 154453), washed twice with PBS, and fixed in 4% formaldehyde in PBS for 15 min at room temperature. Subsequently, the slides were washed with TBS (25 mM Tris, pH 7.4, 100 mM NaCl), and permeabilized for 15 min in TBS containing 0.1% Triton X-100, and washed with TBST (0.05% Tween 20 in TBS). The slides were then blocked for 2 h with 0.5% milk powder in TBST and subsequently incubated with the appropriate combinations of antibodies overnight at 4 °C. After washing with TBST, proximity ligation was performed using Rabbit PLUS and Mouse MINUS Duolink in situ PLA kits (Sigma, DUO94102), according to the manufacturer’s instructions. Subsequently, the slides were dehydrated, air-dried, and embedded in DAPI-containing antifadent mounting medium (VectorLabs, H-1200). PLA signals were imaged by a Zeiss LSM880 confocal microscope system. The PLA-detected proximity (PROX) complexes (red fluorescent dots) intensity per image (40 ×) was quantified by Image J with particle analysis tool.