Total cell extracts were prepared in 1× sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio, China). Cell proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with GAPDH (1:1000, BM3876, Bosterbio, USA), p-PERK, p-IRE1, ATF4, P16 (1:500, bs-3330R, bs-16698R, bs-1531R, bs-20656R, Bioss, China), H3, NRF2 (1:500, D153567, D121053, Sangon Biotech, China), and TNF-α, IL-6, P21, MT1, MT2, HRD1, VCP, P65, p-P65, IKK (1:200, sc-52746, sc-32296, sc-136020, sc-13180, sc-13177, sc-293484, sc-136273, sc-514451, sc-166748, sc-7606, Santa Cruz, USA). Secondary anti-rabbit, anti-mouse antibodies (1:1000, BM2004, BA1001, Bosterbio, USA) conjugated with horseradish peroxidase were used. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence western blotting substrate (Thermo Scientific). Results were analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation, China).
Bm3876
Manufactured by Boster Bio
Sourced in China, United States
BM3876 is a laboratory instrument designed for the detection and measurement of specific molecular targets. It employs advanced technology to provide accurate and reliable results. The core function of this product is to facilitate biological and biochemical analyses in research and diagnostic settings.
Automatically generated - may contain errors
Lab products found in correlation
P erk, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Nuclear and cytoplasm protein extraction kit, by Wanlei (1 mentions)
Sc 52746, by Santa Cruz Biotechnology (1 mentions)
Sc 13177, by Santa Cruz Biotechnology (1 mentions)
Thermo scientific pierce enhanced chemiluminescence western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bs 3330r, by Bioss Antibodies (1 mentions)
D121053, by Sangon (1 mentions)
Sc 32296, by Santa Cruz Biotechnology (1 mentions)
Sc 136020, by Santa Cruz Biotechnology (1 mentions)
Sc 514451, by Santa Cruz Biotechnology (1 mentions)
Sc 166748, by Santa Cruz Biotechnology (1 mentions)
Ba1001, by Boster Bio (1 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
Molecular analyst software, by Bio-Rad (1 mentions)
Ab76956, by Abcam (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
16 protocols using bm3876
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of Liver Tissue
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The liver tissues of mice in each group or mHSCs were homogenized on ice in a lysis buffer, which contained 20 mM Tris-HCl, pH 7.4, 1% SDS, and 1% of protease inhibitor. The investigators resolved the protein extracts on SDS-PAGE. These samples were then transferred onto polyvinylidene difluoride membranes where they underwent incubation with primary antibodies against Runx2 (1 : 1000 dilution; ab76956, Abcam, Cambridge, UK), F4/80 (1 : 600 dilution), RANKL (1 : 600 dilutions; BA1323, Boster Biological Technology, Wuhan, China), and GAPDH (1 : 1000 dilutions; BM3876, Boster Biological Technology, Wuhan, China) overnight at 4°C. The investigators applied the horseradish peroxidase-coupled secondary antibodies (Boster Biological Technology, China) at 1 : 5000 dilutions at room temperature for 90 minutes. The proteins were visualized using a chemiluminescence kit (Millipore, Billerica, MA, USA). Protein expression was quantified by using the Molecular analyst software (Bio-Rad, Marne-la-Coquette, France).
3
Protein Expression Analysis by Western Blot
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Western blotting was performed using antibodies against TRIM3 (ab111840), ESR1 (ab108398), UBC9 (ab75854), SENP1 (ab108981) and SAE1 (ab185552). The membranes were re-probed with an anti-GAPDH (BOSTER, BM3876) as the loading control.
4
Tizoxanide and 3-Methyladenine Interaction
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Tizoxanide (Tiz) (CAS No:173903-47-4) with a purity of over 98% and 3-methyladenine (3-MA) (CAS No:5142-23-4) with a purity of over 95% were obtained from Sigma Aldrich (MO, USA). DMSO was used to dissolve Tiz, 3-MA and applied in equal amounts in each experimental group. Recombinant rat IL-1β (501-RL-010) was procured from R&D systems (MN, USA). Dulbecco's modified Eagle's medium F12 (DMEM/F12), bovine serum albumin (BSA) with a purity of over 98% and fetal bovine serum (FBS) (10099141C) were obtained from Gibco (NY, USA). Antibodies specific for ATG5(#12994), Beclin-1(#3495), LC3Ⅰ/Ⅱ(#12741), COX2(#12282), P-AKT(#4060), AKT(#4685), P-PI3K(#17366), PI3K(#4257), P-mTOR(#5536), mTOR(#2983), ERK(#4695), P-ERK(#4370), JNK(#9258), P-JNK(#9255), P38(#8690), P–P38(#4511) were procured from Cell Signaling Technology (Beverly, MA, USA). Antibody specific for Collagen Ⅱ(15943-1-AP) was supplied from Proteintech Group (Wuhan, China). Antibodies against MMP13 (ab39012), INOS (ab3523) were purchased from Abcam (MA, USA). Antibodies against GAPDH(BM3876) and ADAMTS5(BA3020) were supplied from Boster (Wuhan, China).
5
Western Blot Analysis of Apoptotic Markers
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Western blot analysis was performed on renal lysates or cell extracts using the following primary antibodies: anti-DcR2 (ab108421; Abcam), anti-FLIP (ab8421; Abcam), anti-cleaved caspase 3 (ab214430; Abcam), anti-caspase 8 (ab25901; Abcam), caspase 3 (ab184787; Abcam), anti-caspase 7 (ab255818; Abcam), anti-cleaved caspase 7 (ab256469; Abcam), Akt (sc5298, Santa Cruz), pAkt (sc135650, Santa Cruz), and anti-GAPDH (BM3876; Boster). The intensity of each band was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of Renal Proteins
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Western blotting analysis was performed on renal lysates with RIPA solution containing protease inhibitors (Beyotime Biotechnology, Beijing, China). Protein samples (30 μg) were fractionated by 10–12% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (PVDFs, Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk, these PVDFs were incubated with the following primary antibodies: Acox1 (1:1000, 10957-1-AP; Proteintech group, Wuhan, China), Crot (1:500, ab175450; Abcam, Cambridge, UK), Amacr (1:500, ab268062; Abcam), and anti-GAPDH (1:5000, BM3876; Boster Biotechnology, Wuhan, China) and anti-GAPDH (1:5000, 10494-1-AP; Proteintech group, Wuhan, China). The intensity of each band was analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of EMT Markers
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Western blot analysis was performed as previously described32 (link). The antibodies used in this study were MMP2 (Bioworld, BS1236, 1:500), FN (Novus, NBP1-91258, 1:500), E-Cad (Bioworld, 3195S, 1:500), ALKBH5 (Abcam, ab69325, 1:500), Snail (Bioworld, BS1853, 1:500), ZEB1 (Millipore, ABD53, 1:1000), eEF-1 (Proteintech, 11402–1-AP, 1:500), eEF-2 (Proteintech, 20107–1-AP, 1:500), YTHDF1 (Abcam, ab99080, 1:500), and GAPDH (BOSTER, BM3876, 1:1000). The uncropped gel images are shown in Supplementary Figs 7 and 8 .
8
Quantifying Protein Expression via Western Blot
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EPC cells cultured in six-well plates were transfected with 2 µg plasmid. Twenty-four hours after transfection, cells were lysed by the RIPA (radio immunoprecipitation assay) lysis buffer from beyotime. Then western blot was performed as previously described (Mo et al., 2010 (link)) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. The primary antibodies used for western blot were rabbit anti-Firefly Luciferase (Abcam, #ab185923, 1:5000) and mouse anti-GAPDH (Boster, #BM3876, 1:100). The secondary antibodies were goat anti- rabbit IgG (immunoglobulin) (Boster, #BM3894, 1:5000) and goat anti-mouse IgG (#BM3895, 1:5000; Bosterbio, Pleasanton, CA, USA), respectively. Intensity of the bands in the western blot image was analyzed by the ImageJ software (http://rsb.info.nih.gov/ij/ ).
9
Protein Expression Analysis by Western Blot
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Total proteins extracted from the tissues or cells were subjected to Western blot analysis for HIF‐1α (1:1000 dilution; Abcam, ab51608), ID‐1 (1:1000 dilution; Abcam, ab134163), p53 (1:1000 dilution; Abcam, ab131442), VEGF (1:1000 dilution; Abcam, ab46154), MMP‐9 (1:1000 dilution; Abcam, ab7299) or GAPDH (1:2000 dilution; Boster, BM3876) in standard routine with relative antibodies (Santa Cruz Biotechnology, CA, USA). Quantitative analysis was performed by LAS‐3000 imaging system (FUJIFILM Inc, Tokyo, Japan).
10
Protein Expression Profiling in HepG2 and LM-3 Cells
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HepG2 and LM-3 cells were seeded into 6-well plates and conduct normal culture. Cells are collected when their density reaches 80%. RIPA lysate was used to lyse cells and obtain supernatant by centrifugation. The BCA kit (P0010, Beyotime) was used to determine the protein concentration. Western blotting was performed using antibodies against FDX1 (M05441, Boster), SLC27A5 (A09287-2, Boster), GAPDH (BM3876, Boster). Horseradish peroxidase-labeled Goat anti-rabbit IgG (H+L) (BA1039, Boster) was used as secondary antibodies.
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