Fig 2: Charged multivesicular body protein 2b antibody screening by Western Blot.Lysates of U2OS (WT and CHMP2B KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated Charged multivesicular body protein 2b antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. When the concentration was not indicated by the supplier, which was the case for antibody MA5-21591*, the antibody was tested at 1/1000. Antibody dilution used: MAB7509* at 1/400; MA5-21591* at 1/1000; MA5-36184** at 1/500; ab157208** at 1/1000; 76173** at 1/1000; GTX118181 at 1/1000; GTX109610 at 1/1000; A13410 at 1/500. Predicted band size: 24 kDa. *Monoclonal antibody; **Recombinant antibody.

Fig 3: Charged multivesicular body protein 2b antibody screening by immunofluorescence.U2OS WT and CHMP2B KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated Charged multivesicular body protein 2b antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given to antibodies ab157208** and A13410, which were titrated to 1/1000 and 1/800, respectively, as the signals were too weak when following the suppliers' recommendations. When the concentration was not indicated by the supplier, which was the case for antibodies MA5-21791* and 76173*, we tested antibodies at 1/500. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: MAB7509* at 1/500; MA5-21591* at 1/500; MA5-36184** at 1/1000; ab157208** at 1/100; 76173** at 1/500; GTX118181 at 1/500; GTX109610 at 1/1000; A13410 at 1/800. Bars=10 μm. *Monoclonal antibody; **Recombinant antibody.