FRTL-5 Rat Thyroid Cells Release Thyroglobulin Sequestered in Exosomes: A Possible Novel Mechanism for Thyroglobulin Processing in the Thyroid

Figure 3

SDS-PAGE and Tg Western blot of samples from different stages of exosome isolation by ultracentrifugation: 17,000 ×g supernatant, 200,000 ×g supernatant, and 200,000 ×g (exosome pellet) from samples of 5H (lanes 1–3 and 7–9) and 6H media (lanes 4–6 and 10–12) collected from confluent cultures of FRTL-5 cells incubated for 48 hours. 15 μL samples of resuspended isolated exosomes were loaded for 5H culture 1 and 6H culture 1, while 7 μL samples were loaded for both 5H culture 2 and 6H culture 2. Due to the limited number of lanes in each gel, lanes numbered 1 through 6 and 7 through 12 were run on separate gels. Protein concentrations of resuspended exosome samples were nearly identical in both of the two 5H cultures and both of the 6H cultures. Western blots were treated with a monoclonal anti-human-Tg antibody and developed using SuperSignal West Pico. “Exos” = exosomes (i.e., 200,000 ×g pellet).