Elderberry lectin

SW837 cells were plated until 80% confluence and then lysed in radioimmunoprecipitation assay buffer and protease/phosphatase inhibitors 24 h after treatment, and protein was collected. About 50 μl of SNA-agarose beads (Vector Labs) were washed and then incubated overnight with 500 μg of the collected protein. Sample was pelleted, washed, and resuspended in wash buffer. Protein was removed from the beads using glycoprotein eluting buffer (Vector Labs) or heating in 1× sample buffer with beta-mercaptoethanol. Samples then underwent electrophoresis and protein transfer to polyvinylidene fluoride membranes, membranes were blocked in 5% nonfat dry milk in 1× Tris-buffered saline and 0.1% Tween-20. Membranes were probed for SNA (1:1000) with SNA, elderberry lectin, biotinylated (Vector), and incubated with secondary (1:300) with streptavidin peroxidase. Protein loading was verified using 1:10,000 β-tubulin antibody (Abcam).

Keratinocyte cells were recovered at each time point (T0, 1 and 2 months) and permeabilized with PBS-Triton 0.1% for 4 min at 4 °C. Keratinocyte cells and fingermark samples were saturated in PBS-BSA 1% for 30 min at room temperature. Samples were incubated for 20 min in a humidity chamber using a biotinylated lectin solution (at final concentration 10 µg/ml): Concanavalin A (ConA), Peanut agglutinin (PNA), Elderberry lectin (SNA), and Ulex europaeus agglutinin (UEA) (B-1005, B-1075, B-1305, and B-1065 respectively, Vector Laboratories, USA). After washing with PBS, samples were incubated with Alexa Fluor 555 Streptavidin (S21381, Thermofisher, USA), diluted 1:600, for 20 min in a dark chamber. DNA revelation of the nuclei was done using DNA probes: DAPI or Hoechst 33342 both diluted 1:3000 in PBS-BSA 0.5%. With anti-histone H2B (SAB450223, Sigma-Aldrich, USA), samples were incubated for 2 h (diluted 1/250 in PBS-BSA 0.5%) and then they were incubated with the appropriate fluorescent secondary antibodies for 1 h in the dark (Alexa Fluor 488 conjugated anti-mouse antibody (A11029, Life Technologies, USA)).
Slides were mounted with Prolong mounting medium. Controls without primary antibodies were negative.