HK-2 cells were thoroughly lysed using RIPA lysis buffer. Total protein in the lysate was assessed using Bradford assay. Equal amounts of proteins were loaded onto sodium dodecyl sulphate polyacrylamide gel. The separated samples were then transferred onto a PVDF membrane. The membrane was further blocked using 5% skim milk and incubated with primary antibodies (MCL-1, Abcam, ab32087, 1:1000), PCNA (Abcam, ab29, 1:1500), Cyclin D1 (Abcam, ab16663, 1:1000), BAX (Abcam, ab32503, 1:1000), Bcl-2 (Abcam, ab182858, 1:1000), Cleaved-caspase-3 (Cell signaling, #9664, 1:1500), NOX1 (Abcam, ab121009, 1:1000), NOX2 (Abcam, ab310337, 1:1500), SOD1 (Abcam, ab51254, 1:1000), SOD2 (Abcam, ab68155, 1:1000), Caspase-3 (Abcam, ab184787, 1:1000), β-Actin (Abcam, ab8226, 1:3000), and GAPDH (Abcam, ab8245, 1:2000), overnight at 4 °C. Further, the blots were washed and incubated with secondary antibody for 1 h at RT. Finally, the bands were visualized using ECL western blotting substrate (Invitrogen, 32109, USA). Western blot images were quantified by using ImageJ (V1.8.0.112, National Institutes of Health, Bethesda, MD), with the density of each band normalized to that of β-Actin or GAPDH (Additional file 2 ).
Ab121009
Manufactured by Abcam
Sourced in United States
Ab121009 is a polyclonal antibody that targets the protein ABCB1. It is suitable for use in various applications, including western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Ab166930, by Abcam (2 mentions)
Ab125066, by Abcam (2 mentions)
Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab51254, by Abcam (1 mentions)
Ab68155, by Abcam (1 mentions)
Ab182858, by Cell Signaling Technology (1 mentions)
Ab32087, by Abcam (1 mentions)
Ab184787, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Enhanced chemiluminescence method, by Thermo Fisher Scientific (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab131003, by Abcam (1 mentions)
Ab205236, by Abcam (1 mentions)
Ab3452, by Abcam (1 mentions)
Ab178860, by Abcam (1 mentions)
8 protocols using ab121009
1
Western Blot Analysis of Apoptosis and Oxidative Stress Markers
2
Western Blot Analysis of Inflammatory Markers
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Cells and tissue samples were lysed for protein extraction using RIPA assay. The concentration of each protein sample was determined by a BCA (bicinchoninic acid) kit. Equal amounts (50 mg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, United States). Membranes were blocked using 5% non-fat dry milk with TBST buffer for 1 h, then incubated overnight at 4° C with GPR43 (1:1000, ab131003, Abcam), MFN2 (1:500, ab205236, Abcam), PPARγ (1:1000, ab178860, Abcam), NOX-1 (1:1000, ab121009, Abcam), EBP50 (1:1000, ab3452, Abcam), p47phox (1:1000, ab166930, Abcam), NLRP3 (1:1000, 15101, Cell Signaling Technology, Danvers, MA, US), Caspase-1 (1:1000, sc-392736, Santa Cruz Biotechnology), IL-1β (1:1000, 12242, Cell Signaling Technology, Danvers, MA, US), and β-Actin (1:5000, sc-47778, Santa Cruz Biotechnology). After washing, membranes were probed further with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, Santa Cruz Biotechnology). After washing with TBST for 15 min, immunoreactive bands were exposed by enhanced chemiluminescence method (Thermo Fisher Scientific, Waltham, MA, USA).
3
Immunoprecipitation and Immunoblotting of Succinylated Proteins
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In IP, the lysate protein (500 μg) was mixed with 2 μg protein G agarose antibody (16e266, Millipore, Billerica, MA, USA), and incubated and rotated overnight at 4° C. Proteins were collected as IP production after washing buffer three times. The succinylation signal was detected and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membranes (Millipore, USA), and incubated with primary antibodies: NOX-1 (1:100, ab121009, Abcam), EBP50 (1:100, 3394, Abcam or 1:50, sc-271552, Santa Cruz Biotechnology), p47phox (1:100, ab166930, Abcam or 1:50, sc-17845, Santa Cruz Biotechnology) overnight at 4° C. Membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (sc-2004, sc-2005, 1:2000, Santa Cruz Biotechnology) at room temperature for 1 h. Membrane was developed with ECL (Promega, USA).
4
Western Blot Analysis of Antioxidant Proteins
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After the H9C2 cells in different groups were digested, centrifuged and lysed, the total proteins were isolated for detection of protein concentration. 30 μL of protein samples was taken for electrophoresis in 12% SDS-PAGE gel, electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies overnight at 4°C. The corresponding horseradish-peroxidase-labeled IgG (1:5,000; Abcam, Cambridge, UK) was added on the next day for 1 h before ECL color development. The relative quantity of protein expression in different groups was calculated using Image J software. Primary antibodies used in the study were: Anti – Nrf2 (1:1000; ab62352; Abcam); Anti – SLC7A11 (1:1000; ab175186; Abcam); Anti-ferritin heavy chain (FTH1, 1:1000; ab75972; Abcam); Anti – GPX4 (1:1000; ab125066; Abcam); Anti – FPN1 (1:1000; ab239583; Abcam); Anti – NOX1 (1:1000; ab121009; Abcam); Anti-GAPDH (1: 1000; ab8245; Abcam).
5
Oxidative Stress Signaling in Aorta and HUVEC
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Isolated thoracic aortic tissues or cultured HUVECs were lysed in commercially purchased RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) spiked with 1 mmol/L phenylmethanesulfonyl fluoride (ST506, Beyotime, Shanghai, China) to obtain protein products. The antibodies against the following proteins were used: PXDN (1 μg/mL, ABS1675, Merck, Frankfurter, Germany), RAGE (0.3 μg/mL, ab37647, Abcam, Cambridge, UK), NOX2 (0.25 μg/mL, ab80508, Abcam, Cambridge, UK), NOX1 (0.5 μg/mL, ab131088 and ab121009, Abcam, Cambridge, UK), NOX4 (0.33 μg/mL, ab154244, Abcam, Cambridge, UK), 3-chlorotyrosine (0.1 μg/mL, 3-Cl-Tyr, HP5002, Hycult biotech, Uden, Netherlands), 4-hydroxynonenal (0.43 μg/mL, 4-HNE, ab46545, Abcam, Cambridge, UK), Akt (0.5 μg/mL, SAB4500797, Sigma-Aldrich, USA), p-Akt (0.143 μg/mL, ab18206, Abcam, Cambridge, UK), eNOS (0.25 μg/mL, 61029, BD, Biosciences, NJ, USA) and p-eNOS (0.056 μg/mL dilution, Ser1177, MA5-14957, Invitrogen, CA, USA). Expression of the protein GAPDH (0.5 μg/mL, SAB1405848, Sigma-Aldrich, MO, USA) was used for data normalization. PVDF membranes were incubated with a horseradish peroxidase linked secondary antibody and bands were visualized using gel documentation system (Bio-Rad).
6
Renal Function and Histology Assessment
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In mouse serum, blood urea nitrogen (BUN) and creatinine (Cr) levels were evaluated by GCLabs (Yongin, Korea) using the Cobas 8000 modular analyzer system (Roche, Germany). Kidney tissues from each experimental group were immersion-fixed with 4% paraformaldehyde (pH 7.4) and then embedded in paraffin. Two-micrometer tissue sections were prepared and stained with periodic acid-Schiff (PAS) and Masson’s trichrome using standard protocols for the determination of histological changes and collagen deposition, respectively. Immunohistochemical analysis of kidney tissues detected the Nox-1 (1:100, ab121009, Abcam) and Nox-4 proteins (1:100, MA5-32090, Invitrogen).
7
Protein Expression Analysis in Cells
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Cells were harvested and lysed. Total proteins were collected using the RIPA buffer. The concentration of protein was determined using a BCA kit. The proteins were separated into equal amounts (30 µg) using 12% SDS-PAGE at 120 v. Then, the separated proteins were moved onto PVDF membranes. The membranes were blocked with nonfat milk. The membranes were then incubated with primary antibodies such as anti-TFRC (ab214039, 1:1000, Abcam USA), anti-GPX4 (ab125066, 1:3000, Abcam, USA), anti-NOX1 (ab121009, 1:1000, Abcam, USA), anti-PINK1 (ab216144, 1:1000, Acam, USA), anti-cyto C (ab133504, 1:5000, Abcam USA), anti-Parkin (ab73015, 1:1000, Abcam, USA), anti-Tom20 (ab186735, 1:1000, Abcam, USA), anti-COX II (ab179800, 1:1000, Abcam, USA) and anti-Tubulin (ab6046, 1:500, Abcam, USA) and the next day, with secondary goat-anti-rabbit antibodies (ab6721, 1:2000, Abcam, USA). Subsequently, the bands were visualized using an ECL kit and analyzed using ImageJ v1.52a software.
8
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.
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