Pten regulates endocytic trafficking of cell adhesion and signaling molecules to pattern the retina
Western blot data for Figure 6H and Figure 7E. Uncropped Western blots to examine protein levels in P14 retinal extracellular vesicles from wild-type and Pten conditional-knock-out (cKO) retinas (Figure 6H) and from P0 wild-type and PtencKO retinal lysates (Figure 7E). In Figure 6H, we show that Megf10 protein is detected at lower levels in P14 Pten cKO retinas. In Figure 7E we show that b-catenin levels are elevated in P14 Pten cKO retinas. These data support the hypothesis of altered endocytic/exocytic trafficking of signaling molecules in Pten cKO retinas.
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Retinas were lysed in in NP-40 lysis buffer (0.05 M Tris, pH 7.5, 0.15 M NaCl, 1% NP-40, 0.001 M EDTA) with protease (1X protease inhibitor complete, 1 mM PMSF), proteasome (MG132 at 0.05 mM) and phosphatase (50 mM NaF, 1 mM NaOV) inhibitors. 10 μg of lysate from whole cells or tissue was run on SDS-PAGE gels for western blot analysis as described previously (Ma et al., 2007). To characterize purified EVs, they were boiled at 95oC for 2 mins to break the EV membrane. The protein concentrations were determined using a Micro BCA™ Protein Assay Kit (ThermoFisher Scientific, #23235) following the manufacturer’s instructions. 2 μg of lysate was incubated with the blots overnight at 4°C. Primary antibodies included: β-actin (1:10000, Abcam #8227), Flotillin-1 (1:1000, Cell Signaling #3253), Megf10 (1:1000, Millipore #ABC10), Pten (1:1000, Cell Signaling #9559), ß-Catenin-non-phospho (Active) (Ser33/37/Thr41) (D13A1) (1:1000; Cell Signaling #8814) and TSG101 (1:1000, Sigma #HPA006161). Blots were washed 3 x 10 min in TBST before incubating in secondary antibodies, including either Goat Anti-Rabbit IgG (H+L)-HRP Conjugate (1/50,000; Bio-Rad #1721019) or Goat Anti-Mouse IgG (H+L)-HRP Conjugate (1/50,000; Bio-Rad #1721011). Western blot signals were converted to a chemiluminescent signal using an Amersham ECL Prime Western Blotting Detection Reagent (Cytiva #RPN2232) according to the manufacturer’s instructions. Signal was detected by exposing to autoradiography film (LabForce #1141J52) or using a Bio-rad gel doc and GelCapture MicroChemi 220.127.116.11 software. Each western blot was performed on lysates from three independent retinas, and densitometries were calculated using UN-SCAN-IT gel densitometry software (Silk Scientific). Absolute values (“relative”) or normalized values (over ß-actin) were plotted as indicated.