Cells were lysed in RIPA buffer (50 mM Tris-HCl, at pH 8.0; 150 mM NaCl; 1% (vol/vol) Nonidet P-40; 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche)) on ice. Primary antibodies were used at the following concentrations: USP30 (Santa Cruz, sc-515235, 1:200); TOM20 (Cell Signaling Technology, 42406S, 1:1000); TOM40 (Proteintech, 18409–1-AP, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); TIM23 (Proteintech, 11123–1-AP, 1:500); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); GAPDH (Cell Signaling Technology, 5175S, 1:1000); PINK1 (Cell Signaling Technology, 6946S, 1:1000); ACTIN (Cell Signaling Technology, 3700S, 1:1000). The membranes were incubated with anti-rabbit (LI-COR, 926–32211, 1:15000) or anti-mouse (LI-COR, 926–68072, 1:15000) IgG secondary antibodies for 1 h at room temperature. Images were captured using the Odyssey system (LI-Cor). One representative blot is shown of three independent experiments.
Ndp52
Manufactured by Cell Signaling Technology
NDP52 is a cellular protein that plays a role in the selective autophagy pathway. It functions as an autophagy receptor, recognizing and binding to ubiquitinated cargo targeted for degradation in the autophagosome.
Automatically generated - may contain errors
Lab products found in correlation
Tom20, by Cell Signaling Technology (2 mentions)
Lc3a b, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Odyssey system, by LI COR (2 mentions)
Tim23, by Proteintech (2 mentions)
Parkin, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Tom40, by Proteintech (1 mentions)
Pink1, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Parkin, by Proteintech (1 mentions)
Ripa lysis buffer, by Bioswamp (1 mentions)
Bca protein assay kit, by Bioswamp (1 mentions)
4 protocols using ndp52
1
Immunoblot Analysis of Mitochondrial Proteins
2
Western Blot Analysis of Mitophagy Proteins
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Cells were lysed in RIPA buffer (50 mM Tris-HCl, at pH 8.0; 150 mM NaCl; 1% (vol/vol) Nonidet P-40; 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche)) on ice. Primary antibodies used as described: USP30 (Sino Biological Inc., 14,548-RP01, 1:500); p-AKT (Cell Signaling Technology, 4060S, 1:1000); Parkin (Cell Signaling Technology, 4211S, 1:1000); AKT (Cell Signaling Technology, 4685S, 1:1000); Cleaved PARP (Cell Signaling Technology, 5625S, 1:1000); OPTN (Proteintech, 10837-I-AP, 1:1000); p-mTOR (Cell Signaling Technology, 5536S, 1:1000); mTOR (Cell Signaling Technology, 2983S, 1:1000); TOM20 (Cell Signaling Technology, 42406S, 1:1000); NDP52 (Cell Signaling Technology, 60732S, 1:1000); p-P70S6K (Cell Signaling Technology, 9234P, 1:1000); P70S6K (Cell Signaling Technology, 9202S, 1:1000); LC3A/B (Cell Signaling Technology, 4108S, 1:1000); Beta-Actin (Cell Signaling Technology, 3700S, 1:1000). Secondary antibodies anti-rabbit (LI-COR, 926–32,211, 1:15 000) and anti-mouse (LI-COR, 926–68,072, 1:15 000) IgG were used to incubate membranes at room temperature for 1 h. Images were captured using the Odyssey system (LI-Cor).
3
Protein Expression Analysis of Apoptosis and Autophagy
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Treated cells were lysed with RIPA lysis buffer (Bioswamp), and the protein concentrations in the lysates were measured with a BCA protein assay kit (Bioswamp). Equal amounts of proteins in each sample were loaded onto gels and subjected to SDS-PAGE. The separated proteins on the gels were then transferred to nitrocellulose membranes. Each immunoblot was blocked with 5% nonfat milk in TBST for 2 h prior to an overnight incubation at 4 °C with specific antibodies. The following primary antibodies were used for Western blotting: Bcl-2 (Proteintech, 60178-1-Ig), Bax (Proteintech, 50599-2-Ig), cleaved caspase-3 (Cell Signaling Technology, 9664), LC3B (Bioswamp, MAB43969), Beclin 1 (Proteintech, 66665-1-Ig), Parkin (Proteintech, 14060-1-AP), P62 (Proteintech, 18420-1-AP), Tim23 (Proteintech, 11123-1-AP), CoxIV (Cell Signaling Technology, 4844) and NDP52 (Cell Signaling Technology, 60732). The blots were washed with TBST three times for 15 min each and then incubated with secondary antibodies for 2 h at RT. Finally, enhanced chemiluminescence reagent was used to visualize the bands, and the intensities were determined to further evaluate the levels of each protein.
4
Callyspongiolide Synthesis and Analysis
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pH Pharma acquired an exclusive license from Purdue Research Foundation and synthesized Callyspongiolide at Chem-veda. The primary antibodies of mTOR, p-mTOR s2448, AMPK, p-AMPK, AKT, p-AKT, p-4E-BP t37/46, p-4E-BP t70, 4E-BP, BNIP3, NDP52, parkin, HIF1A, and GAPDH were purchased from Cell Signaling Technology. The primary antibodies of p62 were purchased from BD Biosciences, LC3 from Medical & Biological Laboratories, OXPHOS cocktail, and LAMP2 from Abcam, and β-actin from Santa Cruz Biotechnology.
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