Protein was extracted from T cells using radio-immunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM Na-pyrophosphate, 1 mM β-glycerophosphate, 10% glycerol, protease inhibitor cocktail tablet [Roche, Basel, Switzerland]). Protein lysates (30 μg) were electrophoresed on 13% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with the following antibodies: LC3A/B (1:1,000), SQSTM1/P62 (1:1,000), BECLIN-1 (1:1,000), and ATG12 (D88H11) (1:1,000), followed by HRP-conjugated anti-rabbit IgG (1:3,000). All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific bands on the membrane were visualized with chemiluminescence luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) and developed using the JP-33 automatic X-ray film processor (JPI Healthcare, Seoul, Korea). The target bands were visualized and analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).
Chemiluminescence luminol reagent
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Chemiluminescence luminol reagent is a laboratory product that emits light through a chemical reaction. It is used to detect and quantify the presence of certain enzymes or molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Anti tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Sc 2379, by Santa Cruz Biotechnology (1 mentions)
Hybond p, by Cytiva (1 mentions)
Goat anti mouse igg hrp secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Octa probe d 8, by Santa Cruz Biotechnology (1 mentions)
Octa probe h5, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
4 protocols using chemiluminescence luminol reagent
1
Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of Apoptosis Markers
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HT-29, LoVo wt, control or RAC3, HCT 116 wt, control and shRAC3 cells were harvested and lysed in RIPA buffer with protease inhibitors [29 (link)]. Then, cell lysates were separated via 6% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked in 10% skim milk and incubated with anti-RAC3 (Santa Cruz Biotechnology, USA).
For apoptosis experiments, cells were stimulated with FUra (3.5 μM) or Oxa (0.4 μM) for 6 or 24 h. For Western blot of LC3II/I, cells were pre-incubated with 10 μg/ml E64D and pepstatin A lysosomal protease inhibitors, before incubation with FUra (3.5 μM) or Oxa (0.4 μM) for 90 min. Then, cells were lysed as described before. Samples were separated by 10 or 15% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked in 10% skim milk and incubated with anti-pro-Caspase 3, Beclin 1 and LC3 antibodies.
The anti-Tubulin antibody was used as an internal control (Santa Cruz Biotechnology, USA) in all the assays.
Subsequently, all membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody, and the specific bands were visualized by autoradiography using the chemiluminescence luminol reagent (Santa Cruz Biotechnology, USA).
For apoptosis experiments, cells were stimulated with FUra (3.5 μM) or Oxa (0.4 μM) for 6 or 24 h. For Western blot of LC3II/I, cells were pre-incubated with 10 μg/ml E64D and pepstatin A lysosomal protease inhibitors, before incubation with FUra (3.5 μM) or Oxa (0.4 μM) for 90 min. Then, cells were lysed as described before. Samples were separated by 10 or 15% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked in 10% skim milk and incubated with anti-pro-Caspase 3, Beclin 1 and LC3 antibodies.
The anti-Tubulin antibody was used as an internal control (Santa Cruz Biotechnology, USA) in all the assays.
Subsequently, all membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody, and the specific bands were visualized by autoradiography using the chemiluminescence luminol reagent (Santa Cruz Biotechnology, USA).
3
Quantification of Serum HMGB1 by Western Blot
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Western blot to determine the HMGB1 in sera was done as previously described (22 (link)). Briefly, 1 μL of serum proteins was separated by SDS-PAGE on 12% polyacrylamide gels. Upon protein transfer to a polyvinylidene difluoride membrane (Hybond-P, RPN303F, Amersham Pharmacia Biotech), the membranes were incubated with primary anti-HMGB1 antibody (K-12, Santa Cruz Biotechnology – dilution 1:750) and horse radish peroxidase-conjugated bovine anti-goat secondary antibody (sc-2379; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specific protein bands were visualized using Western Blotting Chemiluminescence Luminol Reagent (sc-2048, Santa Cruz Biotechnology) and Hyperfilm ECL (RPN 3103 K, Santa Cruz Biotechnology). HMGB1 quantification was performed in TotalLab v1.1 (Phoretix).
4
SDS-PAGE and Western Blot Analysis
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Sample preparation for SDS-PAGE was performed by adding Laemmli Sample Buffer. Protein samples were heated at 70 C for 7 min, subjected to 8% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) Immobilon-P membrane (Millipore) by the semi-dry transfer system (Bio-Rad). Membranes were blocked with 5% non-fat dry milk in TBST for 60 min and then incubated with rabbit polyclonal primary antibodies diluted as follows, 1:100 OctA-Probe (D-8), 1:2000 anti-GFP (Santa Cruz Biotechnology), 1:1000 anti-GFP (Cell Signaling Technology) used for immunoblots of Figure S2, 1:3000 anti-D1 (Zhang et al., 2000) , 1:3000 anti-CYTF (Alt et al., 1983) , 1:10000 anti-CYTB6/F (Alt et al., 1983) , 1:3000 anti-LHCP (Adamska et al., 2001) and 1:700 anti-CP (Barbato et al., 1992) . Immunoblotting of co-immunoprecipitation experiments performed with mouse monoclonal OctA-Probe (H-5) (Santa Cruz Biotechnology) in 1:500 dilution. After three washes with TBST, the blots were incubated with 1:10000 diluted goat anti-rabbit IgG-HRP or 1:3000 diluted goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnology). Western blot detection was performed with chemiluminescence luminol reagent (Santa Cruz Biotechnology) by exposure to UltraCruz blue autoradiography film. Western blots were performed in triplicates.
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