In order to remove any exogenous Tf, the media was replaced with DMEM containing no FBS or B27 24 h before the start of experiments. Cells were exposed to apo- or holo-Tf (Sigma, T1147 and T4132) for 10 min and then washed on ice with cold PBS twice. Chilled 100 μl Co-IP lysis buffer (20 mM Tris HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton x-100, and 2 mM EDTA) was added to each well. Cells were collected and incubated with rotation for 30 min at 4 °C. Cell solutions were centrifuged at 14,000×g for 20 min at 4 °C. Supernatant was collected, and protein estimation was performed using Pierce BCA Protein Assay Kit (Thermo, 23227). Approximately 1 mg of protein was used for Co-IP using anti-HA magnetic beads (Thermo, 88837) or Protein G magnetic beads (Thermo, 10003D) complexed with anti-Heph antibody (Santa Cruz, SC-365365) according to manufacturer’s instructions [21 (link)]. Briefly, magnetic beads were washed twice with PBS before adding lysates. The bead and lysate solutions were incubated with rotation for 30 min at room temperature. After washing with PBS, protein was eluted from beads by resuspending in non-reducing sample buffer and boiling at 90 °C for 10 min. Magnet was used to isolate the magnetic beads from the protein solution, which was then reduced using 2 M DTT and then loaded for immunoblotting.
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