Antibodies against the following proteins were purchased: chorein, NBP1-85641 (Novus Biologicals, Centennial, CO, USA); phosphoglycerate kinase 2 (PGK2), 13686-1-AP (Proteintech Group, Rosemont, IL, USA); lactate dehydrogenase C (LDHC), PA5-18779 and acetyl-CoA acetyltransferase 1 (ACAT1), PA5-34676 (Thermo Fisher Scientific, Rockford, IL, USA); isocitrate dehydrogenase 3 subunit alpha (IDH3A), ab118278, annexin A1 (ANXA1), ab214486 and outer dense fiber protein 1 (ODF1), ab197029 (Abcam, Cambridge, UK); translocase of outer membrane 20 kDa subunit (TOM20), sc-11415 (Santa Cruz Biotechnologies, Dallas, TX, USA); alpha-tubulin, 11H10 (Cell Signaling Technology, Danvers, MA, USA); malondialdehyde (MDA), MA5-27559 (Thermo Fisher Scientific).
Ab214486
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab214486 is an antibody product offered by Abcam. It is a primary antibody specifically designed for research applications. The core function of this product is to serve as a tool for the detection and study of target proteins or molecules within biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ma1 80216, by Thermo Fisher Scientific (2 mentions)
Pierce ecl reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab197029, by Abcam (1 mentions)
Nbp1 85641, by Novus Biologicals (1 mentions)
Sc 11415, by Santa Cruz Biotechnology (1 mentions)
Ab131263, by Abcam (1 mentions)
Ab133551, by Abcam (1 mentions)
Ab137040, by Abcam (1 mentions)
Ab210702, by Abcam (1 mentions)
Ab172617, by Abcam (1 mentions)
Ab108306, by Abcam (1 mentions)
Ab137098, by Abcam (1 mentions)
Ab180597, by Abcam (1 mentions)
Ab133348, by Abcam (1 mentions)
Ab52604, by Abcam (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ab245235, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab181602, by Abcam (1 mentions)
17 protocols using ab214486
1
Antibody-Based Protein Detection Protocol
2
Proteomic Profiling of Hippocampal Proteins
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Immunoblots were conducted on hippocampal protein extracts according to our previously described procedure28 (link),35 (link). The primary antibodies used included: Esd (1:10,000; ab133631, Abcam), Idh2 (1:1,000; ab131263, Abcam), Dld (1:10,000; ab133551, Abcam), Sdha (1:1,000; ab137040, Abcam), G6pdx (1:1,000; ab210702, Abcam), Dlat (1:1,000; ab172617, Abcam), Pkm (1:1,000; C103A3, CST), Aldh2 (1:1,000; ab108306, Abcam), Glo1 (1:1,000; ab137098, Abcam), Ogdhl (1:500; 17110–1-AP, Proteintech), Anxal (1:2,000; ab214486, Abcam), Por (1:10,000; ab180597, Abcam), Prdx6 (1:1,000; ab133348, Abcam), Prnp (1:5,000; ab52604, Abcam), and Tpp2 (1:1,000; ab180177, Abcam). Anti-mouse or anti-rabbit horseradish peroxidase-conjugated IgG (1:10,000; Bio-Rad) was used as a secondary antibody. After gel electrophoresis and immunodetection, the protein band intensities were analyzed using Quantity One (Bio-Rad) software. Non-target protein bands in Coomassie blue-stained gels before being transferred to a PVDF membrane were used as the loading control28 (link),36 (link).
3
Extracellular Vesicle Protein Analysis
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Following the lysis and quantification of EVs, samples were loaded with loading buffer and heated at 100°C for 10 min. Equal amounts of protein (40 μg) from each sample were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). The membrane was blocked for 1 h at 25°C using 5% non-fat milk and then incubated overnight at 4°C with gentle rocking in the presence of anti-Annexin A1 antibody (Abcam, ab214486, 1:2000), and anti-CD19 antibody (Abcam, ab245235, 1:1000). The membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were visualized with chemiluminescence using Pierce ECL reagent (Thermo Fisher, USA).
4
Investigating Apoptosis Regulators in Cell Lines
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Antibodies against ANXA1 (ab214486), B-cell lymphoma 2 (BCL-2, ab32124), BCL-2 associated X protein (BAX, ab32503), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab181602) were purchased from Abcam Corporation (Cambridge, MA, United States). Horseradish peroxidase-conjugated donkey anti-rabbit IgG was purchased from Bioworld (Louis Park, MN, United States). The Annexin V-FITC Apoptosis Detection Kit were purchased from Beyotime Biotechnology Technology Company (Shanghai, China). Lipofectamine 3,000 reagent was purchased from Invitrogen Corporation (Carlsbad, CA, United States). siRNA ANXA1 (siANXA1) was purchased from GenePharma Technology Company (Shanghai, China), dissolved in distilled water, and stored at −20°C. siANXA1 sequences was as follows: Forward: 5′ CCUUACCACCAGAAGCUA UTT3′, Reverse: 5′AUAGCUUCUGGUGGUAAGGTT3′.
5
Immunostaining of ANXA1 in Kidney
6
Immunohistochemical Analysis of ANXA1 in Bone
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Tissues were cut into slices with 7-μm thickness, and cryosections were fixed in acetone. Two sections were analyzed per donor. Following blocking in 4% serum, sections were incubated with ANXA1 antibody (Abcam #ab214486; 1:4000) or IgG negative control antibody (Abcam #ab172730; 1:4000) in place of the primary antibody. For immunohistochemistry, sections were then incubated with biotin-labeled secondary antibody (Vector Laboratories, Burlingame, CA), followed by incubation with streptavidin-peroxidase (KPL, Gaithersburg, MD), and incubated in AEC solution (Dako, Lexington, MA). For immunofluorescence, sections were incubated with OsteoSense 680 (1:100), a near-infrared based bisphosphonate calcium tracer, for 1 hour at room temperature, and CNA35-OG488 (1:50), a collagen probe, for 1 hour at room temperature before fixation. ANXA1 antibody incubated slides were incubated with Alexa Fluor 594–labeled secondary antibody (Life Technologies, Carlsbad, CA; 1:200). Immunofluorescence slides were examined using a confocal microscope A1 (Nikon Instruments Inc., Melville, NY), and all images were processed with Elements 3.20 software (Nikon Instruments Inc.).
7
Immunofluorescent Staining of Matrisome Proteins
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Immunofluorescent staining was performed to characterize matrisome protein expression and confirm proteomic profiling results. Frozen sections were used at 3 μm thickness. CryoJane tape was employed to transfer the section to the slide, which were then fixed in paraformaldehyde 4% and washed 2 × 5 min in PBST (phosphate buffered saline with 1% triton X-100). Cryosections where stained with Annexin-A1 (Anxa1, Abcam ab214486, 1:500), Asporin (Aspn, Abcam ab58741, 1:500), Biglycan (Bgn, Abcam ab49701, 1:50), Collagen A1 (III) (Col3a1, Abcam ab6310, 1:200), Lumican (Lum, Abcam ab168348, 1:200), Periostin (Postn, Abcam ab14041, 1:200), Protein S100-A6 (s100a6, Abcam ab244301, 1:50), and Protein S100-A10 (s100a10, Abcam ab187201, 1:50). Primary antibodies were incubated for 2 h, followed by secondary antibodies Alexa Fluor 488 (goat anti-mouse, Invitrogen A21121), Alexa Fluor 488 (goat anti-rabbit, Invitrogen A11008) and Alexa Fluor 555 (goat anti-rabbit, Invitrogen A21422) during 1 h. Stained samples were mounted with Vectashield® (Vector Laboratories H-1500). Imaging was performed with the LSM 700 confocal laser scanning microscope (Zeiss) at 1 Airy Unit (AU) with wavelengths 416, 485, and 555 nm at 200x magnification.
8
Protein expression and localization analysis
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IPZ was obtained was from Calbiochem (Darmstadt, Germany; 401105, 8 μM). Human ABCA1 siRNA was obtained from Thermo Fisher Scientific (Shanghai, China; assay ID 107728, catalog #AM16708).
The following antibodies were used in this study: anti-ABCA1 (ab-18180, Research Resource Identifier [RRID]: AB_444302; Abcam; 1:500 for western blot, 1:200 for IF), anti-ANXA1 (ab-214486; Abcam; 1:2,000 for western blot, 1:1,000 for IF), anti-RBPMS (ABN1362; Millipore; 1:500), anti-β-actin (sc-47778, RRID:AB_2714189 ; Santa Cruz; 1:1,000), anti-importin β (#51186, RRID: AB_2799386 ; Cell Signaling Technology; 1:1,000), anti-beta 1 sodium potassium ATPase (ab-2873, RRID: AB_303375 ; Abcam; 1:250), and anti-histone H3 (#4499, RRID: AB_10544537 ; Cell Signaling Technology; 1:1,000).
The following antibodies were used in this study: anti-ABCA1 (ab-18180, Research Resource Identifier [RRID]: AB_444302; Abcam; 1:500 for western blot, 1:200 for IF), anti-ANXA1 (ab-214486; Abcam; 1:2,000 for western blot, 1:1,000 for IF), anti-RBPMS (ABN1362; Millipore; 1:500), anti-β-actin (sc-47778, RRID:
9
Immunodetection of Annexin, S100A9, Uromodulin, and Osteopontin
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Antibodies specific for annexin A1 (Item No. ab214486), calcium-binding S100A9 (Item No. ab227570), uromodulin (Item No. ab207170), and osteopontin (Item No. ab214050) were obtained from Abcam (Cambridge, UK).
10
Immunohistochemical Antibody Analysis
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The following antibodies were used for tissue immunohistochemistry: rabbit anti-MIST1/BHLHA15 (14896S; Cell Signaling Technologies, Danvers, MA, USA); rabbit anti-alpha smooth muscle actin (ab150301; Abcam); rabbit anti-annexin A1/ANXA1 (ab214486; Abcam); rabbit Sox6 polyclonal antibody (PA5-34616; Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-NDUBB11 polyclonal (YN0924; ImmunoWay Biotechnology, Newark, DE, USA); and mouse anti-FGFR-2 (ab10648; Abcam).
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