The following antibodies were used: anti-PARP (Cell Signaling Technology, 9542), anti-caspase-3 (9661, Cell Signaling Technology, USA), anti-autophagy-related protein 7 (ATG7) (ab133528, Abcam, USA), anti-LC3 (14600-1-AP, Proteintech, USA), anti-p62 (ab56416, Abcam, USA), anti-GAPDH (60004-1-Ig, Proteintech, USA). The following chemical regents were used: PBDE-47 (purity 99.5%, GC/MS) (BDE-047N-3G, AccuStandard Corp, USA), Wortmannin (WM) (S2758, Selleck Chemicals, USA), Rapamycin (RAP) (R5000, Shanghai Haoran, China), Ac-DEVD-CHO (DEVD) (C1206-10 mM, Beyotime Institute of Biotechnology, China). All other chemical regents were analytical grade purchased from credible supplier or as described in the relevant methods.
Ab133528
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab133528 is an antibody product offered by Abcam. It is a purified polyclonal antibody generated in rabbit. The antibody targets a specific protein or antigen, but the detailed function or intended use is not provided in this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Ab108327, by Abcam (5 mentions)
Ab207612, by Abcam (4 mentions)
Ab192890, by Abcam (4 mentions)
Ab109012, by Abcam (4 mentions)
Ab32503, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab56416, by Abcam (2 mentions)
Ab125066, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab48394, by Abcam (2 mentions)
Protein a g, by Thermo Fisher Scientific (2 mentions)
Anti lc3 14600 1 ap, by Proteintech (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Ac devd cho devd, by Beyotime (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Hoechst 33342 b2261, by Merck Group (1 mentions)
17 protocols using ab133528
1
Apoptosis and Autophagy Regulation in PBDE-47 Exposure
2
Thymoquinone-Induced Apoptosis and Autophagy
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Thymoquinone (TQ) was product of MCE (CAS: 490-91-5). Hoechst 33342 (B2261) was bought from Sigma-Aldrich. Cell counting kit‐8 (CCK‐8) was product of Selleck Chemicals (US). N-Acetyl-L-cysteine (NAC) and Z‐VAD‐FMK (ZVF) were obtained from Beyotime Biotechnology (Shanghai, China). The Apoptosis Detection Kit #556547 was purchased from BD (San Jose, CA). Antibodies against Bax (ab32503), Bcl‐2 (ab182858), LC3B (ab192890), Beclin-1 (ab207612), ATG7 (ab133528), MMP2 (ab92536), MMP9 (ab76003) and PD-L1 (ab213524) were bought from Abcam (San Francisco, CA). Antibody against vimentin (V6389) was purchased from Sigma-Aldrich (US). Antibodies against β‐actin (20536‐1‐AP) and Bcl-xl (10783-1-AP) were obtained from Proteintech (Chicago, IL). Antibodies against cleaved caspase 3 (9664), cleaved poly (ADP‐ribose) polymerase (PARP) (5625), E-cadherin (3195), N-cadherin (13116), and SQSTM1/p62 (8025) were obtained from CST company (NJ, US).
3
Western Blot Analysis of Ferroptosis-Related Proteins
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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).
4
Immunohistochemical Analysis of Neuroinflammation
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The rats were anesthetized and perfused with 4% paraformaldehyde, then the brain tissues were collected, fixed and sliced. Next, 10% normal donkey or goat blocking serum (Solarbio, Beijing, China) was used to block brain tissues sections for 30 min at 37°C. The sections (10 μm) were then incubated at 4°C overnight with the following primary antibodies: TSPO (1:100 dilution, pa5-19088; ThermoFish Scientific), Iba1 (1:200 dilution, gb12105; Servicebio), ATG7 (1:100 dilution, ab133528; Abcam), LC3B (1:100 dilution, ab192890; Abcam) and p62 (1:100 dilution, ab109012; Abcam). The secondary antibodies Cy3 conjugated Donkey Anti-Mouse IgG (H+L) (1:200 dilution, gb21401; Servicebio), FITC conjugated Donkey Anti-Goat IgG (H+L) (1:200 dilution, gb22404; Servicebio), Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (H+L) (1:400 dilution, gb25303; Servicebio) and Cy3 conjugated Goat Anti-mouse IgG (H+L) (1:300 dilution, gb21301; Servicebio) were used and incubated 1 h in the dark. The sections were incubated with Hoechst (1:600 dilution, g1011; Servicebio) for 3 min for nuclear staining. The sections were analyzed and photographed using a laser scanning confocal microscope (NIKON Eclipse Ti, Japan). Finally, Image J performs quantitative analysis.
5
Immunoblotting for Autophagy Markers
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Immunoblotting was carried out as described previously [16 (link)]. The following antibodies were employed in this study: Anti-ATG7 (1:2000 dilution), anti-ATG12 (1:1000), LC3 A/B (1:500), and anti-β-actin (1:2000) (cat. ## ab133528, ab155589, ab128025, and ab6276, respectively, Abcam). After blocking in 5% BSA/PBS containing 0.1% of Tween 20 for 1 h, these membranes were probed with the primary antibodies overnight at 4 °C. After incubation with a secondary antibody (1:3000), chemiluminescent signals were registered and scanned, and the band intensity was quantified using ImageJ software. β-actin was used always as the internal loading control.
6
Western Blot Analysis of Cellular Markers
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The protocol of the Western blot was as per the previous study.19 (link) Briefly, total protein was separated using gel electrophoresis and transfected onto a Polyvinylidene Fluoride (PVDF; Millipore, Bedford, MA, USA). The special protein could bind to the primary antibody, followed by incubation with corresponding secondary antibodies. The combination was visualized via an enhanced chemiluminescence kit (Millipore). The primary antibodies were as follows: ATG7 (1:30,000, ab133528, Abcam, Cambridge, MA, USA), Cleaved-caspase-3 (C-caspase-3; 1:500, ab13847, Abcam), Ki-67 (1:5000, ab92742, Abcam), MMP-9 (1:1000, ab38898, Abcam), and GAPDH (1:7000, ab8245, Abcam).
7
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).
8
Immunofluorescence and Immunoblotting Antibody Protocol
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The following primary antibodies were used for immunofluorescence (IF) or immunoblotting (IB): rabbit anti-ATG3 (Abcam, Cambridge, UK; ab108251; IF, 1:50; IB, 1:1000), anti-ATG4B (CST, Leiden, The Netherlands; 5299; IF, 1:100; IB, 1:1000), anti-ATG7 (Abcam, ab133528; IB, 1:1000), anti-SQSTM1 (Abcam, ab109012; IF, 1:200; IB, 1:20,000), anti-OPTN (Abcam, ab23666; IF, 1:50; IB, 1:1000), anti-CACO2/NDP52 (Abcam, ab68588; IF, 1:100; IB, 1:2000), anti-LC3B (CST, 2775; IB, 1:1000), anti-TUBA1A (Santa Cruz Biotechnology, Heidelberg, Germany; SC-5546; IB, 1:1000), anti-USP30 (Abbkine Scientific, Wuhan, China; ABP52679; IB, 1:1000), anti-DNM1L (Cusabio, Houston, TX, USA; CSB-PA002203; IB, 1:1000), mouse anti-PEX14 (IF, 1:100) [40 (link)], and anti-actin (Sigma, A5316; IB, 1:10,000). The secondary antibodies for IF were conjugated to Alexa Fluor 488 (Invitrogen, A11017; 1:2000) or Texas Red (Calbiochem, Darmstadt, Germany; 401355; 1:200), and the secondary antibodies for IB were conjugated to alkaline phosphatase (Sigma, A3687 and A2429; 1:5000 and 1:10,000, respectively).
9
Antibody Profiling for Cellular Mechanisms
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Antibodies against COL1A1 (ab34710), COL4A1 (ab6586), α-SMA (ab5694/ab7817), vimentin (ab92547), p75NTR (ab52987), desmin (ab32362), xCT (ab175186), Ptgs2 (ab179800), GPx4 (ab125066), FTH1 (ab65080/ab183781), FTL (ab69090), Atg5 (ab108327), Atg7 (ab133528), LC3B (ab48394), and 4-HNE (ab46544) were purchased from Abcam Technology (Abcam, Cambridge, UK). Antibodies against FTH1 (sc-376594), FTL (sc-390558), and ubiquitin (sc-8017 AC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against α-SMA (14395-1-AP), Ptgs2 (66351-1-Ig), BECN1 (11306-1-AP), p97/VCP (10736-1-AP), and SQSTM1/p62 (18420-1-AP) were purchased from Proteintech (Proteintech, IL, USA). Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-mouse IgG light chain (AS062), HRP-conjugated goat anti-mouse IgG heavy chain (AS064), and anti-NCOA4 (A5695) were purchased from ABclonal (ABclonal, Wuhan, China). Antibodies against S403-pp62 (#39786), LC3B (#83506), PCNA (#13110), and GAPDH (#5174) were purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA). Monoclonal Anti-FLAG® M2 (F1804), Monoclonal Anti-HA (H9658), and anti-ACTB (A5441) were purchased from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO, USA).
10
Western Blot Analysis of Apoptosis-Related Proteins
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Western blot was performed with standard protocol. Tumor tissues or cells were homogenized using lysis buffer, followed by determining the concentration using bicinchoninic acid kit. A total of 20 μg protein was separated by SDS–PAGE and transferred to the PVDF membranes. Then, the membranes were blocked using 5% nonfat milk for 90 min at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies. The primary antibodies used in this study were: anti‐FBXW7 (ab109617; Abcam), anti‐BCL2‐associated X (BAX) (14796; CST), anti‐BCL2 antagonist killer (BAK) (6947; CST), anti‐MCL‐1 (5453; CST), anti‐BCL2 (3498; CST), anti‐BECN1 (ab207612; Abcam), anti‐Atg7 (ab133528; Abcam), antimicrotubule‐associated protein 1 light chain 3 (LC3) (ab48394; Abcam), and anti‐GAPDH (5174; CST). Then, the membranes were incubated with HRP secondary antibodies for 1 h at room temperature. The signals were visualized using the chemiluminescence (ECL) kit.
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