*4.1. Antibodies*

Fab fragments or full length mAbs raised against CD81 (TS81), CD9 (SYB-1), and CD82 (TS82) were produced, purified and labeled with Atto647N or Cy3B as previously described [13,14]. The three antibodies recognize the extracellular loops of the corresponding tetraspanin [49,50].

#### *4.2. Cell Culture and Treatments*

HB2 mammary epithelial cells isolated from human breast milk were provided by Dr. Fedor Berditchevski and Dr. Elena Odintsova. Different cell lines were used: HB2/Zeo cells weakly expressing CD82 and HB2/CD82 cells overexpressing CD82. All these lines were cultured in a complete DMEM medium containing 10% heat inactivated fetal bovine serum (FBS), 1 mM pyruvate, 10 μg/mL hydrocortisone and 10 μg/mL insulin. Cells in culture were tested for mycoplasma contamination using MycoAlert Mycoplasma Detection kit from Lonza according to manufacturer instructions.

For single molecule tracking experiments, around 10<sup>5</sup> cells were plated 24 h before the experiment in 6-well culture dishes containing 25 mm diameter coverslips that had been previously cleaned using plasma etcher.

For PPMP treatment, cells were grown to a confluence around 50% and D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol·HCl (PPMP) purchased from Matreya was added to reach a final concentration of 2 μM (this concentration was tuned in order to prevent cell death). After one-day treatment, the medium was removed and a fresh medium containing 5 μM of PPMP was added. After two days of culture, the medium was again exchanged for medium containing 10 μM and then routinely cultured as described above. Treatment efficiency was either assessed by flow cytometry using anti-GD1a antibody or by mass spectrometry.
