For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10 μM BAF or 50 nM CQ for 8 h before being lysed by RIPA buffer with PMSF on ice for 15 min. Then the cell lysis was mixed with 5× protein loading buffer and subsequently denaturalized at 100°C for 10 min. Total protein denaturants were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 detergent for 1 h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Cruz), HnRNP-L (ab6106, Abcam), and EGR1 (#4154 CST) at 4°C overnight. Then, all the membranes were immersed in horseradish peroxidase-linked secondary antibodies against rabbit or mouse IgG. Every experiment was carried out in three replicates. The bands were visualized using chemiluminescence imaging system (CLiNX ChemiScope Touch, Shanghai) and quantified by ImageJ software.
Ba2305
Manufactured by Boster Bio
Sourced in China
BA2305 is a microplate reader designed for absorbance-based assays. It can measure optical density in a 96-well microplate format. The product provides accurate and reliable results for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ba0362, by Boster Bio (2 mentions)
Ab6106, by Abcam (1 mentions)
Nb100 2331, by Novus Biologicals (1 mentions)
Sc 28359, by Santa Cruz Biotechnology (1 mentions)
Fluorchem q machine, by Cell Biosciences (1 mentions)
Ba0521, by Boster Bio (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by BioTeke (1 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ab76149, by Abcam (1 mentions)
Ab8805, by Abcam (1 mentions)
Ab0038, by Abways (1 mentions)
β actin, by Boster Bio (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Bs 1074r, by Bioss Antibodies (1 mentions)
Bs 0549r, by Bioss Antibodies (1 mentions)
Bs 0578r, by Bioss Antibodies (1 mentions)
Imagej software, by Merck Group (1 mentions)
10 protocols using ba2305
1
Quantifying Autophagy Markers in Cancer Cells
2
Protein Expression Analysis by Western Blot
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Briefly, whole-cell or tissue lysates were resolved by electrophoresis, and proteins were transferred to nitrocellulose membranes and blotted with antibodies against SPIN1 (1:1000, D152571, Sangon Biotech), ABCB4 (1:500, GTX47122, Genetex), CYP2C8 (1:200, sc-164136, Santa Cruz), UGT2B4 (1:2000, ab173580, Abcam), UGT2B17 (1:1000, abs110602, absin), or β-actin (1:1000, BA2305, Boster). The protein bands were detected using the chemiluminescent substrate with the AlphaView software (Version: 3.2.2.0) on a FluorChem Q machine (Cell Biosciences, Inc., Santa Clara, CA, USA).
3
Western Blot Analysis of Apoptosis Regulators
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Cytoplasm and nucleus extracts were prepared using a Nuclear and Cytoplasmic Protein Extraction Kit (PP2201, Bioteke, China) according to manufacturer's protocol. Whole-cell extract of HCC cells were prepared and subjected to immunoblot analysis. The primary antibodies were rabbit anti-Bcl-2 polyclonal antibody (#2876, CST, diluted 1：1,000), rabbit anti-Bax polyclonal antibody (#2772, CST, diluted 1：1,000), rabbit anti-p53 polyclonal antibody (BA0521, Boster, diluted 1：500), rabbit anti-p53 phospho (pS392) monoclonal antibody (#1644-1, Epitomics, diluted 1：1,000), rabbit anti-Caspase-3 monoclonal antibody (BA2142, Boster, diluted 1：500), rabbit anti-H2b polyclonal antibody (BS1657, Bioworld, diluted 1：500), and β-actin antibody (BA2305, Boster, diluted 1：500). The secondary antibodies, goat anti-mouse IgG-HRP (sc-2005, diluted 1：5,000) and goat anti-rabbit IgG-HRP (sc-2004, diluted 1：5,000), were purchased from Santa Cruz Biotechnology. β-actin served as the loading controls.
4
Comprehensive Western Blot Assay
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Western blot was conducted as previously described.
21 (link) The primary antibodies were YBX1 (1:1000, ab76149; Abcam), YBX2 (1:5000, ab154829; Abcam), PHB2 (1:10000, ab182139; Abcam), HER2 (1:1000, #2165; CST), FAK (1:5000, ET1602‐25; HuaAn Biotechnology), E2F1 (1:1000, #3742; CST), PIN1 (1:2000, 10,495‐1‐AP; Proteintech), AKT (1:1000, ab8805; Abcam), c‐Myc (1:1000, #5605; CST), β‐catenin (1:1000, #8480; CST), Cyclin E1 (1:1000, #20808; CST), p27 (1:1000, #3686; CST), β‐actin (1:1000, BA2305; Boster) or GAPDH (1:5000, AB0038; Abways).
21 (link) The primary antibodies were YBX1 (1:1000, ab76149; Abcam), YBX2 (1:5000, ab154829; Abcam), PHB2 (1:10000, ab182139; Abcam), HER2 (1:1000, #2165; CST), FAK (1:5000, ET1602‐25; HuaAn Biotechnology), E2F1 (1:1000, #3742; CST), PIN1 (1:2000, 10,495‐1‐AP; Proteintech), AKT (1:1000, ab8805; Abcam), c‐Myc (1:1000, #5605; CST), β‐catenin (1:1000, #8480; CST), Cyclin E1 (1:1000, #20808; CST), p27 (1:1000, #3686; CST), β‐actin (1:1000, BA2305; Boster) or GAPDH (1:5000, AB0038; Abways).
5
Quantification of TSPAN13 Protein Expression
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Cells were harvested and protein extracts were obtained with lysis buffer. Equal amounts of protein were electrophoresed on 10% SDS‐PAGE gels, and then transferred to PVDF membrane. After blocking, the membranes were incubated with anti‐TSPAN13 (1:500, abs128170, absin) and anti‐β‐actin (1:1000, BA2305, Boster), followed by incubation with horseradish peroxidase‐conjugated secondary antibodies. Proteins were visualized by using the AlphaView software (Version: 3.2.2.0) on a FluorChem Q machine and analysed by the Multi Gauge V3.2 software.
6
Western Blot Analysis of Cardiac Proteins
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RIPA lysis buffer (catalogue number: P0013C, Beyotime, Haimen, China) was adopted for total protein extraction from mouse hearts or HL‐1 cells. For isolation of nuclear protein, the Nucleoprotein Extraction Kit (catalogue number: C500009, Sangon Biotech, Shanghai, China) was used. After protein concentration quantification, protein samples with equal amount (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skim milk for 1 h at room temperature. The membranes were probed with primary antibodies against ATF4 (bs‐1531R, 1:500, Bioss, Beijing, China), collagen I (bs‐0578R, 1:500, Bioss), collagen III (bs‐0549R, 1:500, Bioss), HIPK2 (bs‐6353R, 1:500, Bioss), Nrf2 (bs‐1074R,1:100, Bioss), HO‐1 (BM4010, 1:500, BOSTER, Wuhan, China), Smurf2 (#12024, 1:1000, Cell Signaling Technology, Trask Lane Danvers, USA), histone H3 (A12477‐2, 1:500, BOSTER), and β‐actin (BA2305, 1:2000, BOSTER) at 4°C overnight. Subsequently, the secondary antibody (#7074, 1:3000, Cell Signaling Technology) was applied for 1 h at room temperature. The immune blots were developed using the enhanced chemiluminescence (Millipore, USA) and subjected to densitometry analysis using ImageJ software (Bethesda, USA). Three independent experiments were conducted.
7
Western Blot Analysis of Macrophage Markers
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Samples were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the protein concentration was measured by the BCA assay (Beyotime, Shanghai, China). Protein samples were separated on 10% SDS-PAGE gels, and the separated proteins were transferred onto nitrocellulose membranes, which were blocked with 5% milk and then incubated overnight with the following antibodies: anti-Alix (1:1,000, ab275377, Abcam, UK), anti-TSG101 (1:1,000, ab125011, Abcam), anti-CD63 (1:1,000, A5271, ABclonal), anti-FoxO1 (1:1,000, C29H4, Cell Signaling Technology), anti-P-FoxO1 (1:1,000, 9461, Cell Signaling Technology), β-actin (1:2,000, BA2305, Boster), anti-iNOS (1:1,000, BA0362, Boster), anti-Arg-1 (1:1,000, 93668, Cell Signaling Technology), anti-P65 (1:2,000, 8242S, Cell Signaling Technology), anti-P-P65 (1:1,000, 93H1, Cell Signaling Technology) and anti-TNF-α (1:1,000, ab205587, Abcam). After incubation for 1 h with the corresponding fluorescent secondary antibody.
8
Protein Expression Analysis in BMDMs
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Total and nuclear proteins were extracted from BMDMs in RIPA buffer containing protease inhibitors (Servicebio, Wuhan, China). Then protein extracts were separated by 10% SDS-PAGE and incubated overnight at 4°C with primary antibodies specific for AhR (BA2013, 96 kDa, 1:200, Boster, China), HIF-1α (PB9253, 97 kDa, 1:1,000, Boster, China), IRF1 (abs118047, 37 kDa, 1:1,000, Absin, China), NF-κB p65 (GB11142-1, 65 kDa, 1:1,000, Servicebio, China), iNOS (BA0362, 130 kDa, 1:200, Boster, China), Arg-1 (GB11285, 40 kDa, 1:5,000, Servicebio, China), H3 (GB13488, 15 kDa, 1:1,000, Servicebio, China), and β-actin (BA2305, 43 kDa, 1:1,000, Boster, China). Blottings were then probed with appropriate secondary antibodies for 2 h at 25°C and assessed via an ECL kit (Millipore, MA, USA). Triplicate analyses of all samples were conducted.
9
Western Blot Analysis of Cell Signaling
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Protein was extracted using RIPA buffer (Beyotime). Then, cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with non-fat milk for 2 h, the membranes were incubated with primary antibodies CDK1 (1:500, BA0027-2, Boster, Wuhan, China), CCND1 (1:2000, PB0403, Boster), γ-H2AX (1:1000, AF5836, Beyotime) and β-actin (1:5000, BA2305, Boster) overnight at 4 °C, followed by incubating with secondary antibody (1:10,000, BA1056, Boster) for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore).
10
Immunoblotting Analysis of Proteins in Transfected CT26 Cells
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The cultured transfected CT26 cells or tissues were collected after being washed with PBS. The total protein was extracted with RIPA lysis buffer (P0013K, Beyotime, China) supplemented with a protease inhibitors cocktail on ice for 30 min. Protein concentration was quantified using the BCA method. An equal amount of protein was separated on 6% or 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE), and then the proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. Skimmed milk (5%) was used to block heterogenetic antigen on the membranes for 1 h. Subsequently, the membrane was incubated with primary antibodies against HK2 (1:500; AF7080, Beyotime, China), LDH (1:500; AF1660, Beyotime, China), AKT, p-AKT Thr308 (1:1000; BS2987 and BS4009, respectively, Bioworld Technology, Co., Ltd., USA), HIF1α (1:1000), GLUT1 (1:500), ART1 (1:1000), and β-actin (1:1000; BA2305, Boster Biological Technology, Ltd., China) at 4°C overnight, followed by incubation with secondary antibodies (1:5000 dilutions) for 30 min at room temperature. Secondary antibodies, peroxidase-conjugated antigoat or rabbit IgG, were then added and incubated for 30 min at room temperature. The protein bands were visualized using the enhanced chemiluminescence detection BeyoECL Plus kit (Beyotime, China), and subsequently imaged and analyzed.
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