Figure 4.

TFR2 shedding is independent of TFR1. (A) HeLa cells were transiently transfected with empty vector (mock) or TFR2^WT-C-FLAG vector. Eighteen hours after transfection media were replaced with serum-free media and cells were treated or not for 24 h with increasing concentrations (0.5, 1.25, 5 μM) of bovine holo-transferrin (B-holo-TF), a ligand specific for TFR2 that does not bind TFR1. Media were collected, concentrated and sTFR2 was pulled down using Sepharose-beads anti-FLAG. Immunoprecipitated proteins were analyzed by western blot using the anti-FLAG antibody. The results were confirmed in three independent experiments and western blot of a representative one is shown. (B) CHO-Trvb-0 (TFR1 deficient) cells were transiently transfected with empty vector (mock) and TFR2^WT-C-FLAG coding vector. Media were replaced 18 h after transfection with serum-free media containing or not 1.25 μM of human holo-TF (H-holo-TF), and surface proteins were biotinylated. Media were collected, concentrated and analyzed by western blot using anti-TFR2 antibody (Abcam). Sn= concentrated media; Lys= total cell lysate, Lys-B = cell lysate pull down with streptavidin-agarose beads. Loading was estimated with anti-β-actin antibody.