Alexa fluor secondary antibody

Briefly, the fixed cells were permeabilized and incubated with anti-PTBP2 antibody, anti-human STAT1 antibody (#10144-2-AP, Proteintech, Chicago, IL, USA), and anti-IRF9 antibody (#14167-1-AP, Proteintech, Chicago, IL, USA) separately. They were further incubated with anti-mouse or rabbit Alexa Fluor secondary antibodies (#4414 or #4409, Cell Signaling Technology, Beverly, USA). The nuclei were subsequently visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining (#P0131, Beyotime, Nanjing, China). Images of the cells were captured by a confocal microscope (Leica SP8, Germany) equipped with a 20× objective and then analyzed using Image-Pro Plus 6.0 software.

PANC-1 cells were harvested and lysed in RIPA buffer containing PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). Following centrifugation at 12,000g for 15min at 4°C, supernatants were collected. Protein concentration was measured by bicinchoninic acid (BCA) assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein samples (80μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Upon completion of electrophoresis, proteins were transferred to PVDF membrane. Membranes were then incubated with 5% skimmed milk in Tris-buffered saline Tween (TBS-T, 20mmol/L Tris, 137mmol/L NaCl, pH 7.6) for overnight at 4°C with primary antibodies. After washed three times with TBS-T buffer, membranes were incubated with Alexa Fluor secondary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), and were then scanned by Odyssey Image System (LI-COR Biosciences, Nebraska, USA). Band intensity was analyzed by Bio-Rad Quantity One Software (version 4.4.0; Bio-Rad, Hercules, CA, USA) for quantifications.

Cells were planted and grown on chamber slides. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Then, slides were blocked with 10% goat serum and incubated with primary antibodies including anti-Rad51 (PC-130, EMD Millipore, 1:500) and anti-BRCA1 (sc-6954, Santa Cruz Biotechnology, 1:200) at 4°C overnight. Then, the slides were extensively washed with PBS and incubated with Alexa Fluor secondary antibodies (1:5000, Cell Signaling Technology) in the dark for 1 h. After washing with PBS and mounting with Prolong Gold antifade DAPI, the slides were assessed using a confocal microscope.

Formaldehyde-fixed cells in optical-bottom multiwell plates were incubated overnight at 4 °C with primary antibodies against e-cadherin and vimentin (Cell Signaling EMT Duplex; Danvers, MA). After washing with PBS, cells were incubated for 1 hour with mouse or rabbit Alexa Fluor secondary antibodies (Cell Signaling; Danvers, MA). Cells were mounted with ProLong Gold Antifade reagent containing DAPI (ThermoFisher Scientific Molecular Probes; Waltham, MA) and imaged after 24 hours using a Zeiss LSM 880 confocal microscope with the same detector settings and excitation laser power settings across groups. Images were analyzed using custom Matlab scripts where fluorescent signal for each protein was normalized to the number of cells based on DAPI-stained nuclei.