Duo92101 1kt

Cells on the glass slides were fixed with 4% paraformaldehyde (PFA) for 20 min and the experiment was performed by following the instructions of the manufacturer of Duolink in situ fluorescence (DUO92101-1KT, Sigma-Aldrich, St Louis, Mosby, United States). Cells of the experimental group were incubated with SOCS2 and ubiquitin or LAMP2 antibodies and cells of the negative group were incubated without antibodies. Mount slides with a minimal volume of Duolink in situ mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min before analyzing in the microscope.

PLA was performed according to the manufacturer’s protocol (Sigma, DUO92101-1KT). CACNG1-Flag was expressed in MIN6 cells for 48 h. Then, the cells were treated with Gal3 (500 ng/ml) for 1 h. Anti-CACNG1 (rabbit; LSBio, LS-C805513, 1:200) and anti-Gal3 (mouse; Abcam, ab2785, 1:200, A3A12) antibodies were used as primary antibodies. Fluorescence images were acquired with a Zeiss LSM 880 microscope.

To detect the association of Sec13 and influenza NS1 by proximity ligation assay (PLA), the Duolink in situ red starter kit was used (DUO92101-1KT—Sigma-Aldrich), following the manufacturer protocol exactly as described to perform this study.

The association between Sox9 and Y14 was assessed using the in-situ proximity ligation assay (PLA) (DUO92101-1KT, Sigma-Aldrich). This method enables the detection and visualization of protein interactions within cells. Ins1 cells were obtained from Dr. Chris Newgard’s lab at Duke University under MTA agreement, plated on glass coverslips, fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then permeabilized in 0.5% Tween in PBS for 30 minutes. Cells were blocked with the blocking solution provided in the PLA kit for 1 hour at 37 °C according to the PLA protocol, followed by incubation overnight at 4 °C with primary antibodies (1:200) against SOX9 and Y14. Duolink PLA probe incubation, ligation, and amplification were then performed according to the manufacturer’s recommendations. Anti-SOX9 (HPA001758, Sigma-Aldrich) and anti-Y14 (05-1511, EMD Millipore) antibodies were used at 1:200 concentration.

HCT116 cells were plated in 12-well Ibidi chambers and cultured for 2 days. Next, the cells were fixed in 4% PFA-PBS and further processed according to the manufacturer’s instructions (DUO92101–1KT, Sigma-Aldrich, Burlington, MA, USA): permeabilization and blocking. The DPLA probe anti-rabbit plus binds to the TKS4 primary antibody (HPA036471, Sigma Aldrich, Burlington, MA, USA, 1:100 dilution), whereas the DPLA probe anti-mouse minus binds to the CD2AP antibody (clone 2A2.1, Millipore, Darmstadt, Germany, 1:250 dilution). The DPLA secondary antibodies generate a signal only when the two DPLA probes interact. Signal detection was conducted with ligation and rolling circle amplification with fluorescently labeled nucleotides. The amplified fluorescent DNA resulted in bright red dots, with each dot representing an individual CD2AP/TKS4 interaction event. Control samples were processed similarly without the addition of the primary antibodies. Staining and image acquisition were performed as previously described [41 (link)]. Briefly, nuclei were visualized via DAPI staining (with DuoLink mounting medium), and images were acquired on a Zeiss LSM710 inverted confocal microscope with a 40× objective (Carl Zeiss). For analysis, 5 pictures were taken from each sample type, control samples did not contain red fluorescent colocalization-indicating dots.