Total protein was extracted with RIPA buffer (Beyotime, China). Next, the concentration of these samples was determined with the BCA method (Beyotime, China). Then, these proteins were separated by the 10% SDS-PAGE gel (Beyotime, China). After that, these proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These membranes were blocked with 5% defatted milk at room temperature for 2 h and incubated with the primary antibodies at 4°C overnight. The primary antibodies used in this research were GPX-4 (ab125066, Abcam), solute carrier family 7 member 11 (SLC7A11; ab37185, Abcam), solute carrier family 3 member 2 (SLC3A2; sc-390154, Santa Cruz), arachidonate-5-lipoxygenase (ALOX5; ab169755, Abcam), autophagy-related 5 (ATG5; ab108327, Abcam), ATG7 (ab133528, Abcam), nuclear receptor coactivator 4 (NCOA4; H00008031-M04, Novus), transferrin receptor (TFR1, ab84036, Abcam), divalent metal transporter 1 (DMT-1; ab222895, Abcam), and GAPDH (ab9485, Abcam). On the second day, these membranes were washed with PBST and incubated with the secondary antibody (Goat anti-rabbit IgG, ab150077, Abcam) for 2 hours. Finally, the bands were developed with enhanced chemiluminescence (ECL) substrates (Millipore, USA).
Ab108327
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab108327 is a primary antibody that recognizes the Phospho-JNK (Thr183/Tyr185) protein. This antibody is suitable for use in Western Blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab207612, by Abcam (17 mentions)
Ab109012, by Abcam (15 mentions)
Ab48394, by Abcam (12 mentions)
Ab192890, by Abcam (9 mentions)
Ab56416, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ab133528, by Abcam (5 mentions)
Ab125066, by Abcam (4 mentions)
Ab51520, by Abcam (4 mentions)
Ab205718, by Abcam (4 mentions)
Ab109268, by Abcam (4 mentions)
Ab32042, by Abcam (4 mentions)
Ab181602, by Abcam (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab32124, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Ab32503, by Abcam (3 mentions)
55 protocols using ab108327
1
Western Blot Analysis of Ferroptosis-Related Proteins
2
Western Blot Analysis of Autophagy Markers
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Cells were rinsed, harvested, lysed and centrifuged. After revolving by SDS‐PAGE, protein samples were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IEVH08100) and incubated in 5% milk TBS‐T for 1 h. The PVDF membranes were first incubated with a primary antibody overnight at 4°C, and then with a secondary antibody conjugating to a fluorescent tag (Invitrogen). The band signals were tested using the Odyssey Infrared Imaging System (LI‐COR, USA). Antibodies against the following antigens were used: OPTN (1:2000, 10837‐1‐AP, Proteintech), LC3 (1:1000, 14600‐1‐AP, Proteintech), Beclin1 (1:5000, 11306‐1‐AP, Proteintech), ATG5 (1:5000, ab108327, Abcam), p62 (1:10,000, ab109012, Abcam), SIRT1 (1:20,000, ab32441, Abcam), AMPK (1:1000, ab207442, Abcam), p‐AMPK (1:1000, #2535T, Cell Signaling Technology) and GAPDH (1:10,000, 60004‐1‐lg, Proteintech). The raw images have been displayed in Figures S13–S17 .
3
Western Blot Analysis of Autophagy Markers
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Tissue samples and cultured cells were lysed with RIPA lysis buffer containing protease inhibitor. SDS-PAGE was used to detect the proteins of interest. Proteins were resolved on SDS gels and were electro-transferred to polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Then, the membranes were blocked with 5% non-fat milk and incubated overnight at 4°C with primary antibodies, including anti-LC3B (#ab48394, 1:500; Abcam), anti-p62 (#ab56416, 1:500; Abcam), anti-ATG5 (#ab108327, 1:500; Abcam), anti-BECLIN1 (#ab55877, 1:500; Abcam), and anti-β-ACTIN (#A8481, 1:4000; Sigma-Aldrich). After washing, the membranes were incubated with a secondary antibody and imaged with Odyssey SA (Gene Company Limited, Hong Kong, China).
4
Quantifying Autophagy Markers in Lung and Tumor Tissues
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For lung cancer patients’ tissue, the sections (5 µm) from the tissue microarray blocks were labelled with antibodies. The LC3B antibody was diluted 1:2000. An HRP- conjugated secondary antibody (Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) was used. The specific target was visualized with a 3, 3’-diaminobenzidine (DAB) detection kit (Beijing Sequoia Jinqiao Biological Technology Co., Ltd.) and counterstained with hematoxylin. Microphotographs from each arrayed tissue were taken with a fixed exposure time and color balance to ensure consistency. LC3B production was quantified using ImagePro Plus9.1 (Media Cybernetic, Silver Spring, MD).
For animal tumor tissue, paraffin sections were first stained with anti-ATG5 (#ab108327, 1:50; Abcam, Cambridge, MA, USA) and anti- LC3B (#ab48394, 1:50; Abcam). Horseradish peroxidase (DAKO, Glostrup, Denmark) was then used as a secondary antibody. When an antigen-antibody-antibody complex was formed, a substrate of the peroxidase, diaminobenzidine, was added as chromogen. The staining was performed according to the manufacturer’s instructions. The pictures were taken at a magnification of 400 × and analyzed using Image Pro-plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Five different pictures were measured for each sample.
For animal tumor tissue, paraffin sections were first stained with anti-ATG5 (#ab108327, 1:50; Abcam, Cambridge, MA, USA) and anti- LC3B (#ab48394, 1:50; Abcam). Horseradish peroxidase (DAKO, Glostrup, Denmark) was then used as a secondary antibody. When an antigen-antibody-antibody complex was formed, a substrate of the peroxidase, diaminobenzidine, was added as chromogen. The staining was performed according to the manufacturer’s instructions. The pictures were taken at a magnification of 400 × and analyzed using Image Pro-plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). Five different pictures were measured for each sample.
5
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.
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.
6
Western Blot Analysis of Apoptosis and Autophagy Markers
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HUVECs were lyzed using RIPA lysis buffer (Beyotime, Shanghai, China) and cell supernatants were collected by centrifugation. The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were isolated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:500), Cleaved caspase-3 (Abcam, ab2302, 1:500), Caspase-3 (Abcam, ab184787, 1:2000), Beclin-1 (Abcam, ab207612, 1:2000), Atg5 (Abcam, ab108327, 1:10,000), p62 (Abcam, ab207305, 1:1000), GRP78 (Abcam, ab108615, 1:10,000), CHOP (Abcam, ab11419, 1:1000), Caspase-12 (Abcam, ab62484, 1:2000), XBP-1 (Abcam, ab220783, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) overnight at 4°C. Membranes were washed in TBST, followed by incubation with secondary antibodies (Abcam, ab205718, 1:50,000; Abcam, ab205719, 1:20,000) for 1 h at room temperature. Protein bots were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China).
7
Western Blot Analysis of Autophagy-Related Proteins
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Treated cells were dissolved in RIPA buffer including protease inhibitors, and then 20 µg protein samples were added to SDS-PAGE gel electrophoresis followed by a transfer of protein to a PVDF membrane. Membranes were sealed using 5% fat-free milk for 1 h and then cultured with antibodies against LOX (Abcam, ab174316), Beclin-1 (Abcam, ab207612), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), ATG5 (Abcam, ab108327), mTOR (Abcam, ab134903), p-mTOR (Abcam, ab109268), AMPKα (Abcam, ab32047), p-AMPKα (Abcam, ab133448), NFATC1 (Abcam, ab25916), ACP5 (Abcam, ab238913), CTSK (Abcam, ab239506), and GAPDH (Cell Signaling Technology, #5174) overnight at 4 °C. After a 30-min washing step in TBS containing 0.1% Tween20, the membranes were cultured with HRP (horseradish peroxidase)-conjugated secondary antibodies (Abcam, ab205718 and ab6728). Visualization was carried out using Pierce™ ECL western blotting substrate (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. Analysis was performed with Image-Pro Plus software.
8
Western Blot Analysis of Autophagy Proteins
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Total protein was extracted from the cells using RIPA lysis buffer. Total proteins were quantified by the BCA protein assay kit (ASPEN). Next proteins (20 μg/lane) were dissolved by 10% SDS-PAGE and then electrotransferred onto a PVDF membrane (Millipore). After that, the membrane was incubated with specific primary antibodies, including anti-Beclin 1 (ab207612, 1:2,000, Abcam), anti-ATG5 (ab108327, 1:1,000, Abcam), anti-ERK (ab184699, 1:10,000, Abcam), anti-p-ERK (ab201015, 1:1,000, Abcam), anti-Bcl-2 (ab32124, 1:1,000, Abcam), anti-cleaved caspase 3 (ab32042, 1:500, Abcam), and anti-β-actin (ab6276, 1:5,000, Abcam), at 4°C overnight. Following incubation with the corresponding secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab7090, 1:5,000, Abcam), the protein blots were developed by ECL reagent. Band intensity was measured using ImageJ software (ImageJ, NIH).
9
Protein Expression Detection by Western Blot
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The total protein of the cell lysates was extracted after treatment with different conditions. A detailed description of the preparation is presented in our previous report [29 (link)]. The primary antibodies used to detect specific proteins were β-actin (A544, Sigma), LC3 (PM036, Medical and Biological Laboratories, Nagoya, Japan), TIMP1 (ab109125, Abcam, Cambridge, UK), Rab37 (LTK BioLaboratories, Taiwan), ATG5 (ab108327, Abcam), and ATG7 (ab133528, Abcam). Samples were incubated overnight at 4 °C. The membranes were incubated with the secondary anti-rabbit (Amersham Pharmacia, Piscataway, NJ, USA) or anti-mouse (Chemicon, Temecula, CA, USA) antibody at room temperature for 1 h. Finally, the membrane was rinsed with enhanced chemiluminescence (ECL) (WBKLS0500; Millipore) and exposed using the BioSpectrum AC system (101-206-009; UVP, Upland, CA, USA).
10
Evaluating Autophagy Markers in Pancreatic Cancer
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Pancreatic cancer cells or xenograft tumor tissues were lysed in ice-cold lysis buffer containing the following reagents: 50 mM Tris–HCl (pH 7.4), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and a complete protease inhibitor cocktail (1 tablet per 10 mL, Roche Diagnostics GmbH, Mannheim, Germany). The total protein concentration was determined using a BCA Protein Assay kit (Beyotime Biotechnology, China). Western blotting was performed using standard methods. The following dilutions were used for each antibody: anti-YY1 (ab109228, Abcam, 1:3000), anti-Beclin 1 (#669922, R&D, 1:1000), anti-ATG5 (ab108327, Abcam, 1:3000), anti-P62 (ab109012, Abcam, 1:10,000), anti-LC3A/B (#12741, Cell Signaling, 1:1000), and anti-GAPDH (SAM1003, Sun Shine Bio, 1:5000). Each blot was independently repeated three times.
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