Optineurin tunes outside-in signaling to regulate lysosome biogenesis and phagocytic clearance in the retina
Blots 1 and 2: Immunoblot of outer segment phagocytosis (rhodopsin) in primary RPE pre-treated or not with 2,4,6-triiodophenol (TIP) (40µM, 30 min or 60 min as indicated) followed by outer segment feeding (~40 OS/cell, 30 min, in the presence of TIP). Loading control: β-tubulin. Lanes 1-3: No treatment, Lanes 4-6: 30 min TIP, Lanes 7-9: 60 min TIP. Blot 3: NativePAGE of RPE lysates immunoblotted for optineurin. Cells were either untreated (Lanes 1-3) or fed with outer segments (~40 OS/cell, 30 min) (Lanes 4-6), or treated with either mitoChlorodinitrobenzoic acid (MC, 5 µM, 30 min) (Lanes 7-9) or Urolithin A (UA, 100 µM, 24 h) (Lanes 10-12). Arrowhead: ~420 kD optineurin hexamers.
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Cells were lysed in 1xHNTG lysis buffer supplemented with protease inhibitor cocktail. Protein concentrations were measured by DC Protein Assay (Bio-Rad). 25 µg lysates were heated at 85℃ for 10 min. Samples were resolved in NuPAGE 4-12% Bis-Tris gel (Invitrogen) at 150V for ~1 h at room temperature. Gels were transferred onto nitrocellulose membranes using the iBlot transfer device (Invitrogen) for 7 min using the preset program 3. The membrane was blocked for 1 h at room temperature in Intercept (PBS) blocker (LI-COR), followed by primary antibody incubation for 16 h at 4℃ (mouse anti-rhodopsin, Millipore MABN15, 1:1000 and rabbit anti-tubulin, Proteintech, 10094-1-AP, 1:1000; primary antibodies diluted in blocker supplemented with 0.1% Tween-20). Signals were detected using IR-Dye secondary antibodies and scanned with Odyssey CLx scanner (LI-COR). For NativePAGE, trypsinized primary porcine RPE monolayers were processed using the NativePAGE sample prep kit (Invitrogen, BN2008). Lysates were resolved in NativePAGE 4-16% Bis-Tris gel (Invitrogen BN1004BOX) and transferred onto PVDF membranes (iBlot, Thermo Fisher). Optineurin oligomers were detected by probing the membrane with rabbit anti-optineurin antibody (Genetex, GTX105447, 1:1000 for 16 h at 4°C). Signal detection were performed as described above. The ladder (NativeMark Unstained Protein Standard, Invitrogen, LC0725) was visualized using Revert Total Protein Stain (LI-COR) according to manufacturer's protocol.