Ubiquitin Proteomics:Article Title: AMBRA1 regulates mitophagy by interacting with ATAD3A and promoting PINK1 stability.
Article Snippet: A pLKO.1 containing a non-target shRNA was used as a negative control (Sigma-Aldrich, SHC016-1EA). .. The primary antibodies used in this study were rabbit antiLC3 (Cell Signaling Technology, 27755), mouse anti SQSTM1/p62 (Santa Cruz Biotechnology, sc-28359; D-3), rabbit anti-AMBRA1 (Millipore, ABC131), mouse anti–multiubiquitin (MBL International, ST1200), rabbit anti-phospho Ser65 ubiquitin (Cell Signaling Technology, 62802), mouse anti-HSP90 (Santa Cruz Biotechnology, sc-13119; F-8), mouse anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (Millipore, CB1001), mouse anti-ACTB/β-actin (C4; Santa Cruz Biotechnology, sc-47778), rabbit anti-PINK1 (Cell Signaling Technology, 6946), mouse-anti PINK1 (8E10.1D6; Novus Biologicals NBP2-36488), rabbit antiLONP1 (Novus, NBP-1-81734), rabbit anti-TOMM20 (Santa Cruz Biotechnology, sc-11415), mouse anti-TOMM20 (F10; Santa Cruz Biotechnology, sc-17764), mouse anti-TOMM70 (Santa Cruz Biotechnology, sc-39054), mouse anti-Ox-PhosII SDHA (Invitrogen, 459200), rabbit anti-ATAD3 (Abnova, H00055210-D01), mouse anti-ATAD3 (Santa Cruz Biotechnology, sc-376185), anti-VDAC/PORIN (MBL, JM3594-100), mouse anti-BCL2L1/BCL-xL (Santa Cruz Biotechnology, sc-8392; H-5), rabbit anti-phospho Ser62 BCL2L1/BCL-xL (Origene, AP01538PU-N), rabbit antiPARP (Cell Signaling Technology, 9542), mouse anti-PRKN /PARKIN (Cell Signaling Technology, 4211) .. Coimmunoprecipitation/immunoblotting experiments were performed by lysing cells in Tris buffer: 10 mM tris, pH 8.0, (Santa Cruz Biotechnology, sc-3715A), 150 mM NaCl (SigmaAldrich, S7653), 10% glycerol (Sigma-Aldrich, G7757), 0.5% NP-40 (Sigma-Aldrich, 56741).
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