Journal: International Journal of Molecular Sciences

Article Title: Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells

doi: 10.3390/ijms22083993

Figure Lengend Snippet: Co-localization of AnxA6, AnxA2, or fetuin-A (FetuA) with TNAP in hFOB 1.19 ( A , C , E ) and Saos-2 ( B , D , F ) cells in resting conditions (R) or after seven-day stimulation with AA and β-GP (S). The cells were incubated with appropriate antibodies: mouse monoclonal anti-AnxA6 ( A , B ), mouse monoclonal anti-AnxA2 ( C , D ), or mouse monoclonal anti-FetuA ( E , F ), all followed by goat anti-mouse IgG-FITC (green); rabbit polyclonal anti-TNAP followed by goat anti-rabbit IgG-TRITC (red) and DAPI for nuclei (blue) and observed under an Axio Observer.Z1 FM (Carl Zeiss, Poznan, Poland) with Phase contrast and appropriate fluorescent filters, magnification 630 x. Arrowheads indicate protein accumulation in vesicular and/or cluster structures. The yellow color and arrows on the merge images indicate AnxA6 ( A , B ), AnxA2 ( C , D ), or FetuA ( E , F ) co-localization with TNAP. Results of a typical experiment are presented.

Article Snippet: The membranes were then incubated with mouse monoclonal anti-annexin A6 (AnxA6; 1:1000, v/v ; BD Transduction Laboratories, Warsaw, Poland), mouse monoclonal anti-annexin A2 (AnxA2; 1:1000, v/v ; BD Transduction Laboratories, Warsaw, Poland), mouse monoclonal anti-fetuin-A (FetuA; 1:500, v/v ; Abcam, Cambridge, UK), rabbit polyclonal anti-TNAP (TNAP; 1:1000, v/v ; Abcam, Cambridge, UK), or mouse monoclonal anti-actin 1 (Actin; 1:2000, v/v ; Abcam, Cambridge, UK) primary antibodies prepared in 3% ( w/v ) milk in TBS supplemented with 0.05% ( v/v ) Tween-20 (TTBS), at 4 °C overnight.

Techniques: Incubation