Western blotting was performed as described previously [20 (link)]. Briefly, the brain protein samples were prepared using Ripa Lysis buffer (Bio-Rad, CA), and equal amounts of protein were run on an SDS-PAGE gel. After being electrophoresed and transferred to a nitrocellulose membrane, the membrane was blocked and incubated with primary antibody overnight at 4 °C. The following primary antibodies were used: AdipoR1 (1:1000, ab126611), p-AMPKα (1:1000, ab133448), AMPKα (1:1000, ab32047), p-NFκB (1:1000, ab86299), NFκB (1:2000, ab16502), tumor necrosis factorα (TNFα) (1:1000, ab6671), interleukin-6 (IL-6) (1:1000, ab6672) (all from Abcam, MA), CTRP9 (1:500, NBP2–46834, Novus, CO), and adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) (1:1000, sc-271,901, Santa Cruz Biotechnology, CA). β-actin was used as an internal loading control. The respective secondary antibodies were incubated for 1 h at room temperature. The bands were probed with an ECL Plus chemiluminescence reagent Kit (Amersham Biosciences, Arlington Heights, PA) and visualized with the image system (Bio-Rad, Versa Doc, model 4000). Relative density of the protein immunoblot images were analyzed by ImageJ software (ImageJ 1.4, NIH, USA).
Ab126611
Manufactured by Abcam
Sourced in United Kingdom
Ab126611 is a lab equipment product offered by Abcam. It serves as a core functional component for laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab16997, by Abcam (2 mentions)
Ab177487, by Abcam (2 mentions)
Ab178847, by Abcam (2 mentions)
Ab178876, by Abcam (2 mentions)
Ab32114, by Abcam (2 mentions)
Ab134175, by Abcam (2 mentions)
Ab2413, by Abcam (2 mentions)
Ab34710, by Abcam (2 mentions)
Ab6672, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab133448, by Abcam (1 mentions)
Ab32047, by Abcam (1 mentions)
Ripa lysis buffer, by Bio-Rad (1 mentions)
Ecl plus chemiluminescence reagent kit, by Cytiva (1 mentions)
Nbp2 46834, by Novus Biologicals (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32358, by Abcam (1 mentions)
Ab23841, by Abcam (1 mentions)
11 protocols using ab126611
1
Western Blotting of Brain Proteins
2
Evaluating AdipoR1 Expression in Ovarian Cancer
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The expression level of AdipoR1 in ovarian cancer tissues was analyzed by immunohistochemical staining. All surgically obtained specimens were preserved in the Pathology Center of the Second Affiliated Hospital, Dalian Medical University for further analysis. We included 15 normal ovarian tissue samples from healthy individuals as controls. Samples were fixed in formalin, embedded in paraffin, and cut into 4-μm sections. For antigen retrieval, tissue sections were treated in a microwave oven with citrate buffer solution (pH 6.0) for 20 min and blocked with normal goat serum for 1 h. After rinsing in PBS 3 times, the slides were incubated for 1 h with anti-human AdipoR1 antibody (ab126611, 1: 100; Abcam, Cambridge, UK) as the primary antibody. A HRP-conjugated goat anti-rabbit IgG H&L (ab6721, 1: 500; Abcam, Cambridge, UK) was used as the secondary antibody. The sections were then immersed in running water and hematoxylin was used for counterstaining.
3
Adiponectin Signaling and Autophagy Regulation
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Antibodies against phospho-AMPK (2531) at Thr172, AMPK (2603), phospho-IKKα (Ser176)/IKKβ (Ser177; 2078S), IKKα (2682), IKKβ (2370), phospho-IκB at Ser32/36 (9246), IκB (4814), phospho-ULK1 at Ser317 (6887), and ULK1 (8054) were from Cell Signaling. Anti-PGC1α (ST1204) was purchased from Millipore. Antibodies against AdipoR1 (ab126611), AdipoR2 (ab77612), UCP1 (ab23841), and C/EBPβ (ab32358) were from Abcam. Anti-LC3B (L7543) from Sigma was used for Western blot, analysis and Anti-LC3 pAb (PM036) from MBL International was used for staining. Anti-adiponectin and anti-β-tubulin were kindly provided by Dr. Lily Dong (University of Texas Health Science Center at San Antonio, San Antonio, TX) as described previously (Alsted et al., 2009 (link); Meng et al., 2017 (link)). AdipoRon (924416–43-3) and 5Z (253863–19-3) were obtained from Cayman Chemical Company. IRAK 1/4 inhibitor I (I5409) and rapamycin (R8781) were from Sigma-Aldrich. 3-MA (189490) was purchased from EMD Millipore. Two recombinant mouse adiponectin (full-length and mutated C39A) proteins (ab62957 and ab94676) were purchased from Abcam. Mouse ST2/IL-1 R4 antibody (MAB10041) was from R&D Systems.
4
Immunohistochemical Analysis of Brain Samples after ICH
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The brain samples used for immunohistochemistry staining were prepared according to a
previously described protocol27 (link). Briefly, animals were deeply anesthetized at 24 h after ICH and transcardially
perfused with 100 ml ice-cold PBS followed by 60 ml of 10% paraformaldehyde. The whole
brain was collected and fixed in 10% paraformaldehyde for 24 h and dehydrated in a 30%
sucrose solution for 3 days. After being kept frozen at –80°C, brains were cut into 8
μm-thick coronal sections on a cryostat (LM3050 S; Leica Microsystems, Bannockburn,
Germany). Immunofluorescence staining was conducted as previously described28 (link). Briefly, brain samples were incubated overnight at 4°C with the primary antibodies
including anti-Iba-1 (1:200, ab178847, Abcam), anti-NeuN (1:200, ab177487, Abcam) and
anti-GFAP (1:200, ab16997, Abcam), and anti-AdipoR1 (1:200, ab126611, Abcam). The
corresponding secondary antibodies (1:500, Jackson Immunoresearch, West Grove, PA, USA)
were added to the brain sections and allowed to incubate at room temperature for 2 h. The
sections were visualized and photographed by using a fluorescence microscope (Leica
Microsystems).
previously described protocol27 (link). Briefly, animals were deeply anesthetized at 24 h after ICH and transcardially
perfused with 100 ml ice-cold PBS followed by 60 ml of 10% paraformaldehyde. The whole
brain was collected and fixed in 10% paraformaldehyde for 24 h and dehydrated in a 30%
sucrose solution for 3 days. After being kept frozen at –80°C, brains were cut into 8
μm-thick coronal sections on a cryostat (LM3050 S; Leica Microsystems, Bannockburn,
Germany). Immunofluorescence staining was conducted as previously described28 (link). Briefly, brain samples were incubated overnight at 4°C with the primary antibodies
including anti-Iba-1 (1:200, ab178847, Abcam), anti-NeuN (1:200, ab177487, Abcam) and
anti-GFAP (1:200, ab16997, Abcam), and anti-AdipoR1 (1:200, ab126611, Abcam). The
corresponding secondary antibodies (1:500, Jackson Immunoresearch, West Grove, PA, USA)
were added to the brain sections and allowed to incubate at room temperature for 2 h. The
sections were visualized and photographed by using a fluorescence microscope (Leica
Microsystems).
5
Antibody-based Signaling Pathway Analysis
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Antibodies against PCNA (2586), phosphor-MEK1/2 (9154), phosphor-p90RSK (11989) and phosphor-MSK1 (9595) were obtained from Cell Signaling Technology (CST, MA, USA). Antibodies against AdipoR1 (ab126611), p90RSK (ab32114), cyclinD1 (ab134175), CDK4 (ab108357), CDK6 (ab124821), total-ERK1/2 (ab184699), phospho-ERK1/ (ab76299) and total-MEK1/2 (ab178876) were obtained from Abcam (Cambridge, UK). Recombinant human adiponectin (1065-AP) and recombinant human epidermal growth factor (EGF, 236-EG) were obtained from R&D System.
6
Adiponectin Signaling Pathway Analysis
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Antibodies of Cleaved-caspase 3 (9661), caspase 3 (9662), PCNA (2586), phospho-MEK1/2 (9154), phospho-p90RSK (11989), phospho-MSK1 (9595), phospho-p38 MAPK (9211), Bax (2774) and Bcl-2 (2872) were obtained from Cell Signaling Technology (CST, MA, USA). Antibodies of AdipoR1 (ab126611), AdipoR2 (ab189446), p90RSK (ab32114), cyclinD1 (ab134175), total-ERK1/2 (ab184699), phospho-ERK1/ (ab76299), total-MEK1/2 (ab178876), phospho-AMPK (ab133448) and phospho-mTOR (ab109268) were obtained from Abcam (Cambridge, UK).
Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System. MEK1/2 inhibitor U0126 was obtained from CST (9911).
Recombinant human adiponectin (1065-AP), recombinant mouse adiponectin (5095-AC) and recombinant human epidermal growth factor (236-EG) were obtained from R&D System. MEK1/2 inhibitor U0126 was obtained from CST (9911).
7
Immunohistochemical Profiling of Lung Adenocarcinoma
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Two lung adenocarcinoma tissues and paired paracarcinoma were used for immunohistochemical staining (IHC). In brief, paraffin-embedded tissue sections were deparaffinized, rehydrated, and pretreated for epitope retrieval. After blocked with 5% goat serum for an hour, the sections were incubated with appropriate primary antibodies overnight at 4 degrees. The primary antibodies used were from Abcam/Santa Cruz/Servicebio/Invitrogen: ADIPOR1 (1:500, ab126611, Abcam), ARRB1 (1:200, ab32099, Abcam), S100A12 (1:20, sc-101347, Santa Cruz), CD1b (1:200, Abcam, ab173576), HAMP (1:50, sc-101347, Santa Cruz), HMOX1 (1:1000, GB11845, Servicebio), KL (1:200, ab181373, Abcam), S100A7 (1:20, sc-52948, Santa Cruz), S100A2 (1:2000, GB111077, Servicebio), VEGFA (1:20, sc-7269, Santa Cruz), VIPR1 (1:50, Invitrogen, PA3-113), and TUBB3 (1:50, sc-80016, Santa Cruz). Following incubation with an HRP-conjugated secondary antibody (1:300, K8002, Dako), the stained sections were reacted with 3,3’-diaminobenzidine and counterstained with hematoxylin.
8
Immunofluorescence Analysis of Brain Cells
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At 24 h following ICH, mice were perfused under deep anesthesia with cold PBS (pH 7.4). Mice were then infused with 10% formalin. Brains were then removed and fixed in formalin at 4 °C for a minimum of 3 days. Samples were then dehydrated with 30% sucrose in PBS (pH 7.4). The frozen coronal slices (8 μm thick) were sectioned in cryostat (CM3050S; Leica Microsystems). Double immunofluorescence staining was performed as previously described [20 (link)]. The primary antibodies included anti-AdipoR1 (1:200, ab126611), anti-Iba-1 (1:200, ab178847), anti-NeuN (1:200, ab177487), and anti-GFAP (1:200, ab16997) (all from Abcam, MA).
9
Western Blot Analysis of Adiponectin Receptors
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Lung tissue (50 mg) was added with 0.5 mL of the protein lysate (Cell Signaling Technology, Danvers, MA, USA), followed by sonication and homogenization. Then, samples were centrifuged at 10,000 × g for 10 min at 4°C to extract the supernatant for the determination of protein concentration (10 µg/µL) and western blotting. Electrophoresis, membrane transfer, and sealing were performed according to conventional procedures. Primary antibodies, including anti-adiponectin receptor 1 (AdipoR1) (ab126611, Abcam, Cambridge, UK), anti-AdipoR2 (ab77612, Abcam), and anti-T-cadherin (ab167407, Abcam), were diluted 1,000 times and incubated overnight. Anti-β-actin antibody (1:1,000, ab8226, Abcam) was used as an internal reference. Then, the membranes were incubated with the secondary antibody at a dilution of 1:10,000. The membranes were placed on a gel imager (Bio-Rad, Hercules, CA, USA) for development and gray-value calculations.
10
Renal Tissue Immunofluorescence Analysis
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We performed IF analysis of AdipoR1(1:100, Abcam, ab126611), p-AMPK (1:200, Abcam, ab23875), LC3B (1:100, Proteintech, 18725-1-AP), FN (1:200, Abcam, ab2413), and Collagen I (1:200, Abcam, ab34710) by using 4% PFA-fixed, paraffin-embedded renal tissue sections (4 μm thick). Apoptotic renal cells were detected using the TUNEL assay with an In-Situ Cell Death Detection Kit (Roche Applied Science, China), in accordance with the manufacturer’s instructions. ROS in renal tissues were determined by oxidative fluorescent dye dihydroethidine (DHE, 1 μM, Invitrogen).
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