Ab120688

Cells were washed five times with serum-free media (DMEM) to remove any traces of serum. Furthermore, cells were incubated with Dynasore (80 μM; Abcam, Ab120192) or Pitstop 2 (15 μM; Abcam, ab120687) in serum-free media for 30 or 20 min, respectively. DMSO was used as a control for Dynasore and Pitstop 2 negative control (Abcam, ab120688). After incubation, drug-containing media was replaced with spike protein (3 μg per 200 μl of media) and further incubated for 5 min to allow internalization. Finally, cells were processed for immunofluorescence labeling.

Clathrin-mediated endocytosis was evaluated by measuring the uptake of transferrin from Human Serum, Alexa Fluor 488 Conjugate (T13342, Thermofisher Scientific). Clathrin-independent endocytosis was measured by intake of mouse monoclonal antibodies directed toward MHC-I (major histocompatibility complex I; clone w6/32, Biolegend) or control mouse monoclonal anti-cytokeratin 14 antibody (ab7800; Abcam; antibodies directed against intracellular target), followed by removal of the unbound antibody by low pH acid wash, fixation, and staining with secondary fluorescein isothiocyanate–labeled goat anti-mouse antibody (115-095; Jackson Laboratories).^{16 (link)} In some experiments, control (Adcontrol) and CLIC4-overexpressing (AdCLIC4) cells were treated with 20 mg/L protein synthesis inhibitor cycloheximide (CAS 66-81-9; Santa Cruz Biotechnology), with or without the endocytosis inhibitor Pitsop2 (20 μmol/L; ab120687; Abcam)^{16 (link)} or Pitstop2-negative control (20 μmol/L; ab120688; Abcam,) for 2 hours. Pitsop2-negative control is chemically related to Pitstop2 but does not block receptor-mediated endocytosis. BMPRII expression was analyzed by Western blotting.