Ab240635

Manufactured by Abcam

Sourced in United States

Ab240635 is an antibody-based reagent for laboratory use. It is designed to detect and measure a specific target molecule in biological samples. The core function of this product is to serve as a research tool for scientific investigations, without making any claims about its intended use or application.

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Lab products found in correlation

7 protocols using ab240635

Immunoprecipitation of Autophagy Proteins

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Cells were lysed in Lysis buffer (Cell Signaling Technology) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific) and total protein concentration determined (Thermo Fisher Scientific). Cell lysates (500 μg) were incubated for 12 h at 4°C either with 5 µg/ml anti-Runx2 (ab236639; Abcam), 5 µg/ml anti-p62 (ab240635; Abcam), or 5 µg/ml anti-LC3 (PM036; MBL international) or 5 µg/ml anti-rabbit IgG (7074; Cell Signaling Technology).
Subsequently the lysates were incubated with 20 µl Protein G magnetic agarose beads (73778; Cell Signaling Technology) for 30 min at room temperature. Protein bound to the beads was washed five times with Lysis buffer, pelleted using a magnetic rack and boiled for 8 min in NuPAGE LDS sample buffer with NuPAGE sample reducing agent (Thermo Fisher Scientific) before analysis by immunoblotting with Runx2 (ab236639; Abcam), p62 (ab240635; Abcam), and LC3 (PM036; MBL international) antibodies as described above.

Phadwal K., Koo E., Jones R.A., Forsythe R.O., Tang K., Tang Q., Corcoran B.M., Caporali A, & MacRae V.E. (2022). Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor. Journal of cellular physiology, 237(11).

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Exosome Protein Profiling via Western Blot

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Total proteins from the exosomes or cells were extracted and detected using the bicinchoninic acid kit. Briefly, 40 µg of total protein was separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (120 V, 90 min) and transferred to polyvinylidene fluoride (PVDF) membranes (90 V, 90 min). For blocking, 5% non-fat milk was added to the PVDF membranes for 1 h. Anti-CD9 (1: 1,000, ab307085, Abcam, Cambridge, MA, USA), anti-CD63 (1: 1,000, ab217345, ab68418), anti-CD81 (1: 1,000, ab109201), anti-SIRT3 (1: 500, ab189860), anti-AMPK (1: 1,000, ab207442), anti-p-AMPK (1: 1,000, ab133448), anti-LC3II/I (1: 1,000, ab48394), anti-P62 (1: 1,000, ab240635), anti-NLRP3 (1: 1,000, ab263899), anti-apoptosis-associated speck-like protein containing a CARD domain (ASC) (1: 1,000, ab70627), anti-IL-1β (1: 1,000, ab283822), anti-iNOS (1:1,000, ab178945), anti-IL-6 (1: 1,000, ab233706), anti-TNF-α (1:1,000, ab183218), anti-Calnexin (1:1000, ab22595) and anti-GAPDH (1:1,000, ab8245) were added and incubated overnight at 4 °C. Moreover, the horseradish peroxidase-labeled goat-anti-rabbit secondary antibody (1:5,000 diluted) was incubated at 25 ℃ for 2 h. Protein blots were detected using Pierce™ ECL (Thermo Fisher, Waltham, USA) in ChemiDoc MP (Bio-Rad, California, USA).

Yang L., Liu S., He Y., Gan L., Ni Q., Dai A., Mu C., Liu Q., Chen H., Lu H, & Sun R. (2024). Exosomes regulate SIRT3-related autophagy by delivering miR-421 to regulate macrophage polarization and participate in OSA-related NAFLD. Journal of Translational Medicine, 22, 475.

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Western Blot Analysis of Mitochondrial Regulators

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H9c2 cells and myocardial tissues were lysed with RIPA buffer (Thermo Fisher Scientific), total proteins were extracted from the lysis solution. Applying 10% SDS-PAGE gel electrophoresis, protein samples were separated. The separated proteins were transferred to PVDF membranes, and then incubated sequentially with primary antibodies and secondary antibody. The antibodies used in western blotting shown as follows: rabbit anti-FbxL4 (1:1000; bs-13166R; Bioss, Beijing, China), rabbit anti-PINK1 (1:500; 23274-1-AP; Proteintech, Wuhan, China), mouse anti-Parkin (1:500; 39-0900; Thermo Fisher Scientific), rabbit anti-LC3 (1:1000; 14600-1-AP; Proteintech), rabbit anti-cleaved caspase-3 (1:1000; PA5-114687; Thermo Fisher Scientific), rabbit anti-p62 (1:1000; ab240635; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:1000; ab196495; Abcam), rabbit anti-Mfn2 (1:1000; ab124773; Abcam), rabbit anti-Drp1 (1:1000; ab184247; Abcam), rabbit anti-GAPDH (1:10000; ab181602; Abcam), rabbit anti-β-actin (1:1000; ab8227; Abcam), goat anti-mouse IgG (1:5000; ab6789; Abcam), goat anti-rabbit IgG (1:10000; ab6721; Abcam). The immunoreactive bands were visualized by enhanced chemiluminescence reagent.

Wu R., Xu F., Li J., Wang F., Chen N., Wang X, & Chen Q. (2024). Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1. iScience, 27(2), 108982.

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Western Blot Analysis of Cellular Proteins

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The protein concentration in the cell or tissue lysates was measured using a BCA kit (Beyotime) to determine the loading volume. Proteins were mixed with loading buffer (Beyotime), denatured in a boiling water bath for 3 min, separated by electrophoresis (80 V for 30 min and 120 V for 12 h), and transferred onto membranes. Protein transfer was performed in an ice bath at 300 mA for 60 min, followed by 1–2 min of membrane washing. Next, the membranes were soaked in blocking solution for 1 h at normal temperature and incubated with primary antibodies against β-actin (ab8227, 1:2500), METTL14 (ab300104, 1:1000), LC3 (ab128025, 1:1000), Beclin-1 (ab62557, 1:1000), p62 (ab240635, 1:1000), Bax (ab182733, 1:2000), Bcl-2 (ab196495, 1:1000; Abcam), cleaved caspase-3 (9661, 1:1000, Cell signaling Technology), or UBR1 (sc-515753, 1:500, Santa Cruz Biotechnology) on a shaker for 1 h at normal temperature. After washing for 3 × 10 min, the membrane was transferred to a secondary antibody solution (IgG, 1:10,000, Abcam) and incubated for 1 h at room temperature. After color development, the blots were visualized with a chemiluminescence imaging system (Bio-Rad).

Wang C., Zhu X., Chen R., Zhang X, & Lian N. (2023). Upregulation of UBR1 m6A Methylation by METTL14 Inhibits Autophagy in Spinal Cord Injury. eNeuro, 10(6), ENEURO.0338-22.2023.

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Western Blot Analysis of Autophagy Markers

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The proteins were extracted from SH-SY5Y and SK-N-SH cells using Radioimmunoprecipitation(RIPA) lysis buffer (Beyotime, Biotechnology, Shanghai, China). A Bicinchoninic acid (BCA) Assay Kit (Beyotime Biotechnology) was used to measure the concentration of total protein and proteins were separated by SDS-PAGE. Afterwards, the separated proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with skimmed milk (5%) for 1 h, primary antibodies p62 (1:1000, #ab240635, abcam), Beclin-1 (1:2000, ab207612, abcam), LC3B (1:2000, #ab192890, abcam) and β-actin (1:1000, #ab8226, abcam) were performed to incubate the PVDF membranes overnight at 4 °C. Subsequently, secondary antibodies were used to incubate the membranes for 1 h, and an ECL kit (Thermo Fisher Scientific) was used to visualize the protein bands. Image J software (NIH-Image, Bethesda, MD, USA) was applied to analyze the gray values.

Shen D.F., Qi H.P., Zhang W.N, & Sang W.X. (2023). Resveratrol Promotes Autophagy to Improve neuronal Injury in Parkinson’s Disease by Regulating SNHG1/miR-128-3p/SNCA Axis. Brain Sciences, 13(8), 1124.

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Streptozotocin-Induced Diabetes Model

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Glucose, streptozotocin (STZ), citrate buffer, dimethylsulphoxide (DMSO) and collagenase type II were purchased from Sigma‐Aldrich. Rapamycin was acquired from MedChemExpress LLC. Cell culture reagents were obtained from Gibco. Cell counting kit‐8 (CCK‐8) was purchased from Dojindo. Glucometer was obtained from Yuwell. The primary antibody against GAPDH (60004‐1‐Ig) was purchased from Proteintech, primary antibodies against p21WAF1 (ab86696), p62 (ab240635), LC3 (ab62721), and Atg7 (ab223365) were acquired from Abcam, and primary antibodies against HuR (#12582), p53 (#2524), p‐p53 (#9284), p16INK4A (#80772) and LC3‐II (#2775) were purchased from Cell Signaling Technology. Goat anti‐rabbit and anti‐mouse IgG‐HRP antibodies were purchased from Bioworld. In Jackson ImmunoResearch, Alexa Fluor^® 594‐ and 488‐conjugated secondary antibodies were obtained. In Beyotime, 4′,6‐Diamidino‐2‐phenylindole (DAPI) was purchased.

Shao Z., Ni L., Hu S., Xu T., Meftah Z., Yu Z., Tian N., Wu Y., Sun L., Wu A., Pan Z., Chen L., Gao W., Zhou Y., Zhang X, & Wang X. (2020). RNA‐binding protein HuR suppresses senescence through Atg7 mediated autophagy activation in diabetic intervertebral disc degeneration. Cell Proliferation, 54(2), e12975.

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Western Blot Analysis of NP Cell Signaling

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NP cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (NCM Biotech). The following antibodies were used for western blotting: rabbit monoclonal antibodies: (GAPDH, 1:1000 dilution, #5174, CST; p62, 1:1000 dilution, ab240635, Abcam; FNDC5, 1:1000 dilution, ab174833, Abcam; p-AMPK, 1:1000 dilution, #2535, CST; AMPK, 1:1000 dilution, #5832, CST; p-mTOR, 1:1000 dilution, #5536, CST; mTOR, 1:1000 dilution, #2983, CST); rabbit polyclonal antibodies: (cleaved-caspase3 1:1000 dilution, #9661, CST; p16INK4a 1:1000 dilution, A0262, ABclonal); mouse monoclonal anti-LC3B (1:1000 dilution, #83506; CST).

Zhou W., Shi Y., Wang H., Chen L., Yu C., Zhang X., Yang L., Zhang X, & Wu A. (2022). Exercise-induced FNDC5/irisin protects nucleus pulposus cells against senescence and apoptosis by activating autophagy. Experimental & Molecular Medicine, 54(7), 1038-1048.

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