Treated HepG2 cells were collected and lysed with RIPA buffer containing a protease inhibitor cocktail (Sigma, USA), and the concentration of protein was determined by a BCA kit (Beyotime, China). Extracted proteins were separated in 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA) followed by incubation with the following primary antibodies: anti-TRAF6 (rabbit, ab137452, Abcam), anti-MAPK11 (rabbit, ab137066, Abcam), and anti-GAPDH (rabbit, ab181602, Abcam).
Ab137452
Manufactured by Abcam
Sourced in United States
Ab137452 is a lab equipment product offered by Abcam. It is designed to perform specific laboratory functions. The core function of this product is to [CORE_FUNCTION]. Further details on the intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab181602, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab90522, by Abcam (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab68418, by Abcam (1 mentions)
Ab226977, by Abcam (1 mentions)
Ab223052, by Abcam (1 mentions)
Ab104156, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab37168, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab32518, by Abcam (1 mentions)
Chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using ab137452
1
Protein Expression Analysis in HepG2 Cells
2
Antibody Selection for Protein Analysis
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Abs specific for Actin (I-19) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The antibodies to TRAF6 (ab137452) and GroEL (ab90522) were purchased from Abcam (Cambridge, UK). The antibody to GST (91G1), HAUSP (D17C6) and K48 Ub (D9D5) were purchased from Cell Signaling Technology (Danvers, MA, USA) and Flag (M185-3L), His (D291-3), and HA (M180-3) were purchased from MBL life science (Sunnyvale, CA, USA). P22077 (S7133) were purchased from Sellekchem (Houston, TX, USA). siHAUSP (sc-77373) were purchased from Santa cruz Biotechnology. RNAiMAX (13778) were purchased from Invitrogen (Waltham, MA, USA). shHAUSP were purchased from Horizon discovery (Waterbeach, UK).
3
Western Blot Analysis of Extracellular Vesicle Proteins
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The extraction of total protein was done utilizing RIPA buffer (Beyotime) and quantified utilizing the BCA protein assay kit (Tiangen, Beijing, China). An equal amount of protein was resolved with sodium dodecyl sulfonate-polyacrylamide gel (Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (Sigma-Aldrich). After blocking in 5% defatted milk for 1 h at indoor temperature, the membranes were incubated with primary antibodies against CD9 (ab223052; Abcam, Cambridge, MA, USA), CD63 (ab68418; Abcam), CyclinD1 (ab226977; Abcam), Bax (ab104156; Abcam), matrix metalloprotein 13 (MMP13; ab39012; Abcam), aggrecan (ab36861; Abcam), TRAF6 (ab137452; Abcam) or GAPDH (ab37168; Abcam) overnight at 4 °C and indicated secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. The ECL kit (Beyotime) was employed for chemiluminescence imaging.
4
Western Blot Analysis of Kidney Injury Markers
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Both kidney tissue and HK-2 cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail. Protein concentrations were measured using a BCA protein assay kit (Invitrogen, USA). Protein samples in 10% sodium dodecyl sulfate-polyacrylamide difluoride (SDS-PAGE) membranes were separated and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, MA). After blocking by 5% non-fat milk at RT for 1 h, membranes were incubated with primary antibodies against Hsp70.1 antibody (1:1000; PA5-97846, ThermoFisher), HSP70 (1:1000; ab79852, Abcam), TRAF6 (1:1000; ab137452, Abcam), GAPDH (1:1000; 9485, Abcam), IκBα (1:1000; ab32518, Abcam), p-IκBα (1:1000; ab133462, Abcam), p65 (1:1000; ab32536, Abcam), p-p65 (1:1000; ab222494, Abcam), TNF-α (1:1000; ab183218, Abcam), IL-6 (1:1000; ab259341, Abcam), Bax (1:1000; ab32503, Abcam), Bcl2 (1:1000; ab194583, Abcam), caspase 3 (1:1000; ab184787, Abcam) overnight at 4 °C. After washing with Tris-Buffered Saline Tween-20 (TBST) buffer on the next day, the membranes were incubated in secondary antibodies (Goat Anti-Rabbit IgG, 1:5000, ab205718, Abcam) at RT for 2 h. At last, protein bands were treated with chemiluminescence detection kit (Thermo Fisher Scientific, USA) and detected by Fusion-capture system (Fusion, France).
5
Investigating HSP70-TRAF6 Interaction in Cells
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HEK293T cells were transfected with the plasmids of FLAG-HSP70 and HA-TRAF6 (Public Protein/Plasmid Library, Nanjing, China). After 24 h, cells were harvested with RIPA buffer and incubated with 1 μg anti-HA antibody (ab1424, Abcam) and 20 μL Protein A/G beads (Santa Cruz Biotechnology, USA) overnight at 4 °C. To detect the endogenous interaction of HSP70 and TRAF6, HK-2 cells were lysed and incubated with 3 μg anti-IgG (ab172730, Abcam) or anti-HSP70 (ab5442, Abcam) antibody and 20 μL Protein A/G beads overnight at 4 °C. Proteins were analyzed by Western blotting followed by an anti-HA (1:1000) and anti-FLAG (1:1000; F7425, Sigma) antibody. Endogenous proteins were detected using anti-TRAF6 antibody (1:1000; ab137452, Abcam).
6
Immunohistochemical Analysis of Bone Markers
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After antigen retrieval and normal goat serum blocking, the sections of rat alveolar bone tissues were incubated at 4 °C overnight with rabbit anti-mouse primary antibodies against RUNX2 (SAB1403638, 1:500, Sigma-Aldrich, St. Louis, MO, USA), OCN (AB10911, 1:500, Sigma-Aldrich), IRAK1 (SAB4504245, 1:200, Sigma-Aldrich, St. Louis, MO, USA), and TRAF6 (ab137452, 1:100, Abcam). The next day, the sections were incubated with goat anti-rabbit IgG (ab6721, 1:1000, Abcam) at 37 °C for 20 min and then with horseradish peroxidase-labeled streptomyces ovalbumin working solution (Imunbio, Beijing, China) at 37 °C for 20 min, followed by DAB (Whiga, Guangzhou, China) color development. Hematoxylin (Shanghai Bogoo Biological Technology Co., Ltd., Shanghai, China) was applied for counterstaining the sections. Images were finally observed and photographed under a microscope.
7
Western Blot Analysis of Betaine's Effects
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Western blot analysis was performed to analyze the influence of betaine treatment on the MAPK pathway, ROS-related enzymes and osteoclastogenesis-related markers. We used M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, United States) to extract total protein from BMMCs. Twenty micrograms of protein sample per lane was loaded onto (11%) SDS-PAGE gels. When the protein sample reached the bottom of the gel, the power was cut off, electrophoresis was stopped, and the protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, 1620177). The primary antibodies included anti-P38 (Abcam, 1:1,000, ab170099), anti-P-P38 (Abcam, 1:1,000, ab195049), anti-ERK (Abcam, 1:1,000, ab17942), anti-P-ERK (Abcam, 1:1,000, ab201025), anti-JNK (Abcam, 1:1,000, ab179461), anti-P-JNK (Abcam, 1:1,000, ab124956), anti-TRAF6 (Abcam, 1:1,000, ab137452), anti-Nox1 (Abcam, 1:1,000, ab121009), anti-HO1 (Abcam, 1:1,000, ab137749), anti-catalase (Abcam, 1:1,000, ab217793), anti-TRAP (Abcam, 1:5,000, ab52750), anti-MMP9 (Abcam, 1:1,000, ab228402), and anti-Cathepsin K (Abcam, 1:1,000, ab187647), followed by the secondary antibody (Biosharp, 1:5,000, BL003A). The results were probed by chemiluminescence.
8
Protein Expression Analysis of AR42J Cells
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According to the instructions, cerulein-treated AR42J acinar cells with RIPA buffer (Beyotime) and 30 μg denatured proteins were subjected to detect protein expression. Membrane with protein attached was incubated with primary antibodies against TRAF6 (1:1000, AB137452, Abcam), TLR9 (1 : 200, ab134368, Abcam), NLRP3 (1 : 1000, ab260017, Abcam), caspase-1 (1 : 1000, ab179515, Abcam), ASC (1 : 1000, ab283684, Abcam), and GSDMD (1 : 1000, ab210070, Abcam). The loading control was GAPDH (1 : 10 000, ab181602, Abcam). Band analysis software is ImageJ (version 1.8.0_172).
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