Fig 2: Myoferlin is expressed in exosomes from breast and pancreas cancer cell linesA. Western blot analysis of myoferlin expression in exosomes isolated from multiple breast and pancreas cancer cell lines. CD9 and FLOT1 were used as positive controls for exosomes whereas the GRP78 (ER) and COX1 (mitochondria) were employed as controls to exclude presence of contaminating organelles. B. Immunogold electron microscopy of exosome preparations from MDA-MB-231 cells stained for myoferlin. (A-B) Representative images of three independent experiments are shown.

Fig 3: Photomicrograph representing immunocytochemical staining for flotillin-1 (FLOT1) in human endometrial epithelial cells (hEEC). Primary hEEC and the cell lines ECC-1 were treated with diluent or IL11 (100 ng/ml) for 24 h. Cytospins were prepared and subjected to immunocytochemistry as described in the Materials & Methods. Positive staining for FLOT1 showed as brown pigment with blue nuclear counterstain. (A, D) Negative IgG (B) Control primary EEC (C) IL11 100 ng/ml primary hEEC (E) Control ECC-1 (F) IL11 100 ng/ml ECC-1. Bars represent 200 μm.

Fig 4: Photomicrograph representing immunohistochemical staining for flotillin-1 (FLOT1) in human endometrial tissue throughout the menstrual cycle and in ECC-1 cells. Positive staining for FLOT1 showed as brown pigment with blue nuclear counterstain. (A) Menstrual, d1-4 (B) Proliferative, d5-14 (C) Early-secretory, d15-20 (D-F) Mid-secretory, d21-24 (G-H) Late-secretory, d25-d27. (GE) Glandular epithelial, (LE) Luminal epithelial. Insets in each case are matched negative controls. Positive control of human tonsil included (I). Bars represent 200 μm. Semi-quantitative analysis of staining intensity for FLOT1 in GE (J). Data represented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, Kruskal Wallis test, with Dunn's post-hoc test.

Fig 5: Identification of Cbl interactors with and without EGF stimulation using SILAC MS. A, the SILAC MS scheme shows that HeLa cell proteins were labeled with stable isotopes to compare Cbl complexes without EGF (light, L) and with 30 min of 100 ng/ml EGF stimulation (heavy, H). Cbl was immunoprecipitated (IP) from differentially treated cell lysates and the IPs were mixed at 1:1 ratio. The mixed IP was either directly used to generate peptides for MS (1) or was separated by SDS-PAGE (2), divided into fractions as indicated, and each fraction was analyzed separately with MS. B, HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting Cbl/Cbl-b (positive control), FLOT1, or FLOT2 and seeded on 96-well plate. Phospho-EGFR ELISA was used to detect the levels of EGFR phosphorylation at Y1173 upon 100 ng/ml EGF stimulation for indicated time periods. The pEGFR Y1173 signal was normalized to the Janus-green whole-cell staining signal, and the average values for three experiments (±SEM) were plotted versus EGF stimulation time. C, HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting FLOT1, FLOT2 separately and together for 48 h and then treated with 25 ng/ml EGF for the indicated time periods. The lysates were subjected to Western blot analysis with the indicated antibodies. D, EGFR phosphorylation upon EGF stimulation is shown as an average of pEGFR/EGFR ± SEM calculated by densitometry analysis of Western blot (as shown in D) for three independent experiments, with a.u indicating arbitrary units. E, degradation of EGFR upon EGF stimulation was calculated by densitometry analysis of Western blot in D from seven independent experiments and plotted as an average of EGFR/HSC70 ± SEM. EGFR levels in untreated samples (0 EGF) were set as 1, with a.u indicating arbitrary units. Asterisk (*) denotes p < 0.05, (**) indicates p < 0.01 as compared with the corresponding time points for siNeg using Student’s t test. MW in kDa is shown to the left of the Western blot panels. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SILAC MS stable isotope labeling with amino acids in cell culture mass spectrometry