Ab52472

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab52472 is a lab equipment product manufactured by Abcam. It is designed for use in scientific research applications.

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10 protocols using ab52472

NLRP1 Activation and Autophagy Signaling

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The NLRP1 activator muramyl dipeptide (MDP) (Selleck, USA) was then added at 100 μmol for 24 hours to the oxidative stress model according to the preliminary experiment and the published studies [21 (link)–24 (link)]. The cell viability determination was the same as that in Section 2.1.
Total protein from HTR-8/SVneo cells was prepared with RIPA lysing buffer. The sample proteins (20 μg) of the different groups were separated using 10% SDS-PAGE and transferred onto PVDF membranes. These membranes were incubated with a primary antibody, followed by incubation of the anti-rabbit IgG secondary antibody. Protein expression was detected with an enhanced chemiluminescence detection kit. GAPDH served as an internal control. The antibodies included those for IL-1β (Abcam, ab216995), pro-IL-1β (Abcam, ab216995), pro-CASP1 (Abcam, ab207802), CASP1 (Abcam, ab207802), Beclin-1 (Abcam, ab207612), LC3 (Abcam, ab51520), p62 (Abcam, ab211324), ATG5 (Abcam, ab108327), ATG7 (Abcam, ab52472), NLRP3 (Abcam, ab263899), NLRP1 (BioLegend, 679802), and GAPDH (Abcam, ab128915). An ECL chemiluminescent reagent and imaging system (Bio-Rad, USA) were used to display protein bands, with the collected images analyzed using Bio-Rad software.

Li M., Sun T., Wu X., An P., Wu X, & Dang H. (2021). Autophagy in the HTR-8/SVneo Cell Oxidative Stress Model Is Associated with the NLRP1 Inflammasome. Oxidative Medicine and Cellular Longevity, 2021, 2353504.

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Autophagy Pathway Protein Expression

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The expressions of ALKBH5, ATG7 (Autophagy related 7), ATG5(Autophagy related 5), Beclin1, ULK1 (Serine/Threonine-Protein Kinase ULK1), PI3KC3, p-MTOR (Mammalian target of rapamycin), and Actin proteins were analyzed by WB. The primary antibodies included monoclonal rabbit anti-ALKBH5 (Abcam, ab234528), rabbit monoclonal to ATG7 (Abcam, ab52472), rabbit monoclonal to ATG5 (Abcam, ab109490), rabbit polyclonal to Beclin1 (Abcam, ab62557), rabbit polyclonal to ULK1 (Abcam, ab203207), rabbit monoclonal to PI3KC3 (Abcam, ab40776), rabbit monoclonal to p-mTOR (Abcam, ab109268). Monoclonal mouse anti-β-actin (Abcam, ab8226) was used as an internal control to evaluate the band density on a gel imaging system.

Zhu H., Gan X., Jiang X., Diao S., Wu H, & Hu J. (2019). ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2. Journal of Experimental & Clinical Cancer Research : CR, 38, 163.

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Quantitative Immunohistochemical Analysis of Autophagy Markers

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The slides were incubated with the primary antibodies (1:100 dilution, rabbit monoclonal antibody (mAb) for ATG7, ab 52472, Abcam, UK; 1:500 dilution, rabbit mAb for LC3, 3868 CST, USA; 1:200 dilution, rabbit mAb for Beclin-1, 3395 CST, USA; and 1:75 dilution, ki-67, clone MIB-1, Dako, Santa Clara, CA, US) overnight at 4 °C and with Anti-rabbit Envision+/horseradish peroxidase (HRP) (Dako) or Anti-mouse Envision+/HRP (Dako) secondary antibody for 1 h. Staining of ATG-7, LC3, and Beclin-1 was categorised semi-quantitatively based on the percentage of positive tumour cells: 0 (5% positive cells), 1 (6–25% positive cells), 2 (26–50% positive cells), 3 (51–75% positive cells), and 4 (>75% positive cells). Cytoplasmic and membrane staining intensities were also determined semi-quantitatively as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Sections’ scores were defined as the extent of staining × intensity, while the Ki-67 score was calculated as the percentage of positively stained cells among the total number of malignant cells scored. The scoring was conducted using three high-power (×400) fields in the invasive edge of the tumour, which represents the spectrum of staining observed in the whole section.

Liu Y., Baba Y., Ishimoto T., Tsutsuki H., Zhang T., Nomoto D., Okadome K., Yamamura K., Harada K., Eto K., Hiyoshi Y., Iwatsuki M., Nagai Y., Iwagami S., Miyamoto Y., Yoshida N., Komohara Y., Ohmuraya M., Wang X., Ajani J.A., Sawa T, & Baba H. (2020). Fusobacterium nucleatum confers chemoresistance by modulating autophagy in oesophageal squamous cell carcinoma. British Journal of Cancer, 124(5), 963-974.

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Western Blot Analysis of Autophagy Proteins

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The cells were harvested, lysed using ice-cold NP-40 lysis buffer (Cell Signaling Technology), centrifuged and the supernatant collected. Equal amounts of the supernatant protein (50 μg) were loaded onto an SDS-PAGE gel. The samples were electrophoresed and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA). WB analysis of cell lysates [28 (link),29 (link)] was carried out using the following primary antibodies: SOCS5 (ab244384, 1:2000), Bcl-2 (ab182858, 1;1000), p62 (ab280086, 1:2000), LC3B (ab221794, 1:2000), Beclin 1 (ab207612, 1:2000), ATG7 (ab52472, 1:2000), Bcl-2 (phospho S70, ab218123, 1:2000), cleaved Caspase-3 (ab32042, 1:3000), cleaved Caspase-9 (ab2324, 1:3000), and GAPDH (ab9485, 1:1000), which were purchased from Abcam. After the membranes were washed three times for 10 min with Tween-Tris-buffered saline buffer, they were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 120 min. The positive signal for the target protein was analyzed using a Tanon 2500 image analyzer (Shanghai, China). Densitometry of specific blotted bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/), and the intensity values were normalized against the GAPDH loading control.

Yu J., Han L., Yang F., Zhao M., Zhou H, & Hu L. (2022). SOCS5 contributes to temozolomide resistance in glioblastoma by regulating Bcl-2-mediated autophagy. Bioengineered, 13(6), 14125-14137.

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Notch Signaling Pathway Protein Detection

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Samples were run by SDS-PAGE and transferred to low autofluorescence polyvinylidene difluoride membrane (Immobilon, FL, Millipore). Primary antibodies were used at the concentration described below overnight at 4 °C in blocking solution (1% milk powder in PBS). Secondary antibody (IR-Dye conjugation goat anti-mouse or -rabbit LI-COR Biosciences) was used at 1:50,000 dilution in blocking solution for 1 h. The membrane was scanned on the LI-COR Odyssey (LI-COR Biosciences) using the Image Studio software.
Dilution of primary antibodies. 1:250: Notch1 (ab52627, Abcam), NICD (ab8925, Abcam), Hes1(ab71559, Abcam), Dll1 (ab76655, Abcam), ATG7 (ab52472, Abcam), Numb (ab14140, Abcam)
1:1,000: LC3 (NB 100-2220, Novus), ATG16L1 (pAb PM040, MBL), Beclin (#3738S, Cell Signalling), VAMP3 (gift from A.A. Peden)
1:2,000: Actin (A2066,Sigma-Aldrich).
Notch1 on western blot shows Notch NTMD (125 kDa). It is the form which is cleaved during maturation at the plasma membrane. The NICD (activated Notch1) antibody detects VLLSRKRRRQHGQC, a sequence, which is not accessible in the uncleaved form. It is exposed after S1 cleavage. The protein is detected at 80 kDa.

Wu X., Fleming A., Ricketts T., Pavel M., Virgin H., Menzies F.M, & Rubinsztein D.C. (2016). Autophagy regulates Notch degradation and modulates stem cell development and neurogenesis. Nature Communications, 7, 10533.

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Autophagy and copper regulation in cells

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The following antibodies against GCLM (1:2000; ab126704), p62 (1:2000; ab109012), ATG5 (1:3000; ab109409), ATG7 (1:10,000; ab52472), LC3B (1:3000; ab192890), p-mTOR (1:2000; ab109268) were purchased from Abcam (Cambridge, UK). The following antibodies against BIP (1:1000; 3177), mTOR (1:1000; 2983), p-AMPK α (1:2000; 2573S), AMPKα (1:2000; 5831), Beclin1 (1:1000; 3495) were purchased from Cell Signaling Technology (Danvers, MA). The CD68 (1:200; 28058-1-AP) and HO-1 (1:1000; 10701-1AP) was purchased from Proteintech (Chicago, USA). The ULK1 (1:1000; A5149) was purchased from Bimake (Houston, USA). The β-actin Ab was obtained from Bioss (1:5000; bs-0061R) (Beijing, China). The QuantiChrom^TM Copper Assay Kit (DICU-250) was purchased from Bioassay (California, USA).

Xiao J., Tu B., Zhou X., Jiang X., Xu G., Zhang J., Qin X., Sumayyah G., Fan J., Wang B., Chen C, & Zou Z. (2021). Autophagy deficiency exacerbates acute lung injury induced by copper oxide nanoparticles. Journal of Nanobiotechnology, 19, 162.

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Quantitative Analysis of miRNA and Autophagy Markers

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For quantitative real-time PCR (RT-PCR) analysis, total RNA from cells was isolated using RNA simple Total RNA Kit (Tiangen Biotech), and miRNAs were performed using the miRcute miRNA isolation kit (Tiangen Biotech) according to the manufacturer’s instructions. RT-PCR was performed using Hairpin-it™ miRNAs RT-PCR Quantitation Kit (GenePharma, China) and samples were amplified for 40 cycles as follows: 95°C for 12 s, 62°C for 40 s, and 72°C for 30 s. The miR-106a expression was calculated relative to U6 snRNA. For Western blotting, proteins were loaded onto 12 or 15% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (PVDF). Membranes were blocked in 5% non-fat milk in PBST for 1 h, and incubated with anti-ULK1(Abcam, ab167139), anti-ATG16L1 (Abcam, ab188642), anti-ATG7 (Abcam, ab52472), anti-LC3 (Abcam, ab51520), and anti-GAPDH (Abcam, ab245355). Immunoreactive band was performed using ECL reagent (Amersham Pharmacia) and quantified by using Image J software (NIH).

Liu K., Hong D., Zhang F., Li X., He M., Han X., Zhang G., Xu G., Stonehouse N.J., Jiang Z., An W, & Guo L. (2021). MicroRNA-106a Inhibits Autophagy Process and Antimicrobial Responses by Targeting ULK1, ATG7, and ATG16L1 During Mycobacterial Infection. Frontiers in Immunology, 11, 610021.

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Western Blot Analysis of Autophagy Markers

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RIPA lysate (Beyotime, Shanghai, China) was used to obtain total proteins, 100 μg of which were segregated with SDS‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. TBST containing 5% skim milk was used for membrane incubation for 1 hour. Then, the membranes experienced incubation with primary antibodies including anti‐SMA (Sigma‐Aldrich), anti‐Apg7 (ab52472, 1/100 000, Abcam, Cambridge, MA, USA), anti‐APG5L/ATG5 (ab108327, 1/1000, Abcam), anti‐LC3A/B (ab128025, Abcam), anti‐SQSTM1/p62 (ab56416, Abcam) and anti‐GAPDH (ab181603, 1:10 000, Abcam) at 4°C overnight. The membranes were washed in TBST 3 times and incubated with anti‐rabbit IgG H&L (HRP) secondary antibody (ab6721, 1:2000, Abcam) at room temperature for 1.5 hours. After washed with TBST thrice, the membranes were subjected to colour reaction by ECL Plus from Life Technology, and GAPDH was detected as control groups.

Yu S., Dong B., Fang Z., Hu X., Tang L, & Zhou S. (2018). Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR‐204‐3p and inhibiting autophagy. Journal of Cellular and Molecular Medicine, 22(10), 4886-4898.

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Protein Extraction and Western Blot Analysis

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Total protein from the cells was extracted using radio-immunoprecipitation assay cell lysis buffer (Cell Signaling Technology) supplemented with the protease inhibitor (Roche Ltd, Basel, Switzerland). The protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following a 1-h block in 5% non-fat milk at room temperature, the membranes were incubated with the primary antibodies against KDM5C (1:1,000, ab264168, Abcam), PFDN5 (1:1,000, ab129116, Abcam), ATG7 (1:1,000, ab52472, Abcam), P62 (1:1,000; NBP1-48320, Novus Biologicals, Littleton, CO, USA), Beclin1 (1:1,000, #3495, Cell Signaling Technology), LC3 (1:1,000; #3868, Cell Signaling Technology), Vimentin (1:1,000, NBP1-31327, Novus Biologicals), N-cadherin (1:1,000, NBP2-19457, Novus Biologicals), GAPDH (1:1,000; ab181602, Abcam) at 4 °C overnight. Subsequently, the membranes were incubated with the HRP-conjugated IgG (1:20,000, ab6721, Abcam) at room temperature for 2 h. Protein blots were developed using enhanced chemiluminescence reagent and analyzed using Image J.

Yu F., Li L., Gu Y., Wang S., Zhou L., Cheng X., Jiang H., Huang Y., Zhang Y., Qian W., Li X, & Liu Z. (2024). Lysine demethylase 5C inhibits transcription of prefoldin subunit 5 to activate c-Myc signal transduction and colorectal cancer progression. Molecular Medicine, 30, 9.

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Autophagy and Apoptosis Signaling Pathways

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Our team performed western blot analysis employing antibodies to LC3A/B (#12741), total AKT (#4691), phosphorylated AKT (#4060), phosphorylated-mTOR (#2972), total mTOR (#2971), phosphatidylinositol 3-kinase (PI3K) (#4249), cleaved caspase-3 (#9662), and cleaved poly (ADP-ribose) polymerase (PARP) (#94885) from Cell Signaling Technology (Danvers, MA, USA), and p62 (ab109012), ATG7 (ab52472), TCP1 alpha (ab109126), and GAPDH (ab181602) from Abcam (Cambridge, UK), following the protocols.

Chen X., Chen X., Huang Y., Lin J., Wu Y, & Chen Y. (2021). TCP1 increases drug resistance in acute myeloid leukemia by suppressing autophagy via activating AKT/mTOR signaling. Cell Death & Disease, 12(11), 1058.

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