EpoR internalization is through a clathrin-dependent pathway. (A) γ2A cells expressing HA-EpoR with JAK2 or JAK2KD were seeded on glass coverslips. Cell-surface HA-EpoRs were labeled with anti-HA antibodies before Epo induction. Seven minutes after induction, cells were fixed and immunostained with anticlathrin antibodies followed by appropriate fluorescence-conjugated secondary antibodies. Representative confocal images (single section) are presented. Selected areas of colocalization are indicated with arrows (original magnification ×40; Leica TCS SP5). (B) Knockdown of the clathrin heavy chain abolished ligand-induced EpoR internalization. γ2A cells were transfected with siRNAs to the clathrin heavy chain, and surface EpoR was analyzed by flow cytometry. (C) Knockdown of the clathrin heavy chain abolished Epo-induced EpoR degradation. (D) Knockdown efficiency of the clathrin heavy chain shown by immunoblotting. Immunoblotting with actin was shown as a control.