Samples were lysed by a radio immunoprecipitation lysis buffer and their protein contents were determined by an Enhanced BCA Protein Assay Kit (Beyotime, China). After centrifugation, the lysates were run by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto the polyvinylidene fluoride membranes. Then, the membranes were blocked for 1 h at 25°C with 5% non-fat dried milk in TBST, incubated overnight at 4°C with primary antibody. The membranes were subsequently incubated with the HRP-conjugated secondary antibody (dilution 1:2,000; Abcam). Protein bands were detected with enhanced chemiluminescence reagents (Amersham). The primary antibodies used are as follows: Bax (ab32503, Abcam), Bcl-2 (ab196495, Abcam), ADAMTS-4 (ab185722, Abcam), ADAMTS-5 (ab41037, Abcam), MMP-13 (ab39012, Abcam), aggrecan (13880-1-AP, Proteintech), Type II collagen (ab34712, Abcam), and FOXO3 (ab23683, Abcam). GAPDH (#5174, Cell Signaling Technology) was used as controls.
Ab23683
Manufactured by Abcam
Sourced in United States, China
Ab23683 is a laboratory equipment product from Abcam. It is a core tool for researchers in various scientific fields. The detailed specifications and technical details of this product are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab81283, by Abcam (2 mentions)
Ab47285, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Sphk1, by Cell Signaling Technology (2 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Ab34712, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Ab185722, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ab49822, by Abcam (1 mentions)
Enhanced chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using ab23683
1
Western Blot Analysis of Cartilage-Related Proteins
2
Western Blot Analysis of Signaling Pathways
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Cells were lysed using a modified radioimmunoprecipitation assay (RIPA, (Beyotime, China) lysis buffer on ice, and the BCA Protein Assay Kit (Beyotime, China) was used to measure the protein concentration. Equivalent amounts of proteins (40 μg per lane) were separated by 10% SDS-PAGE, and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). After that, the membrane was incubated with primary antibodies at 4°C overnight. Antibodies were diluted to 1:1000 for p-Akt (Abcam; ab81283), Akt (Abcam; ab179463), p-FOXO3a (Abcam; ab154786), FOXO3a (Abcam; ab23683), cleaved caspase 3 (Abcam; ab49822), β-actin (Abcam; ab6276). Later on, the membrane was incubated with secondary antibodies (Abcam; ab150077) for 1 h at room temperature. Subsequently, the bands were imaged by an enhanced chemiluminescent substrate kit (Thermo Fisher Scientific).
3
Western Blot Analysis of CTCF and FoxO Proteins
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Samples containing 20 μg of total protein were separated on 8%‐12% SDS‐PAGE gels according to the different molecular weights and then transferred onto nitrocellulose membranes (Whatman, Germany) in transfer buffer using a Mini Trans‐Blot Cell (Bio‐Rad) at 400 mA for 2 hours. The membranes were blocked by incubating them in 5% nonfat milk in TBS‐T for 1 hour at room temperature. Proteins were detected using specific rabbit polyclonal anti‐CTCF (1:1000, ab203312; Abcam, USA), rabbit polyclonal anti‐pFoxO1a (1:200, ab131339; Abcam), rabbit polyclonal anti‐FoxO1a (1:500, ab70382; Abcam), rabbit polyclonal anti‐pFoxO3a (1 μg/mL, ab47285; Abcam), rabbit polyclonal anti‐FoxO3a (5 μg/mL, ab23683; Abcam), rabbit polyclonal anti‐Lamin B1 (1 μg/mL, ab65986; Abcam) and rabbit polyclonal anti‐GAPDH (5 μg/mL, ab9485; Abcam) antibodies. After being washed with TBS‐T, the membranes were incubated with goat anti‐rabbit immunoglobulin G secondary antibodies (1:50 000, ab205718; Abcam) in TBS‐T containing 5% nonfat milk for 45 minutes at room temperature. The grey value of bands was analysed by enhanced ImageJ (Version 1.48u; MD, USA).
4
Protein Expression Analysis via Western Blot
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Cell lysis was performed in RIPA buffer (Beyotime, Nantong, China) containing protease and phosphatase inhibitors (Beyotime). A BCA Protein Assay kit (Beyotime) was utilized to identify protein concentration, and the samples (40 µg proteins per lane) underwent SDS-PAGE with 10% gel for separation. Next, proteins were electrotransferred onto a PVDF membrane (Beyotime) that was sealed by 5% BSA (Beyotime) for 1 h at indoor temperature. Later, we incubated the membrane with primary antibodies against TSG101 (1:1,000, ab125011, Abcam, Shanghai, China), CD63 (1:1,000, ab217345, Abcam, Shanghai, China), FOXO3 (1:1,000, ab23683, Abcam, Shanghai, China), and GAPDH (1:1,000, ab8245, Abcam, Shanghai, China) at 4°C nightlong, and subsequently with secondary antibodies coupled to HRP (Beyotime, Nantong, China) at indoor temperature for 1 h. Immobilon ECL substrate (Millipore) was used to generate signals, which were detected using the Optimax X-ray Film Processor provided by Protec (Shanghai, China).
5
Immunofluorescence Analysis of Muscle Tissue
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After blocking with 10% goat serum in PBS overnight, the paraformaldehyde‐fixed paraffin‐embedded muscle sections were incubated with anti‐CD31 (#SAB5700639, Sigma‐Aldrich) or anti‐laminin (#NB300‐144, Novusbio, Shanghai, China), or anti‐p16 (#PA5‐20379, Invitrogen) or anti‐FOXO3a antibodies (#ab23683, Abcam) in muscle transverse sections overnight at 4°C. After washed three times with 0.1% PBS with Tween 20 (PBST), these sections were incubated with FTIC‐conjugated secondary antibodies (#111‐095‐003, Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 647 (#ab150083, Abcam) for 1 h, respectively. Then, the slides were washed three times with PBST and stained with 1 μg/mL of 4,6‐diamidino‐2‐phenylindole (DAPI) for 30 min to detect nuclei. Images were acquired using a Zeiss Axioskop 2 plus fluorescence microscope. ImageJ software was used to quantify the capillaries, the capillary‐to‐fibre ratio and the expression of p16.
6
Bone Tissue Protein Extraction and Western Blot Analysis
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Total protein was extracted from bone tissues using the bone tissue protein extraction kit (BestBio, China) following the manufacturer's instructions. Concentrations of proteins were measured using a BCA protein assay kit (Thermo Fisher Scientific, IL, USA). For western blot analysis, proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Subsequently, PVDF membranes carrying target peptides were blocked in 5% skim milk (Beyotime, China) and then incubated with primary antibodies against RANKL (1 : 1,000; PA5-110268, Invitrogen, CA, USA), OPG (1 : 1,000; ab73400, Abcam, UK), FoxO3a (1 : 1,000; ab23683, Abcam), Wnt1 (1 : 3,000; PA5-85217, Invitrogen), β-catenin (1 : 5,000; ab73400, Abcam), and GAPDH (1 : 10,000; ab181602, Abcam) at 4°C overnight. Next, membranes were incubated with secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1 : 2,000; ab6721, Abcam) for 2 h. Band intensity of protein peptide was detected using the enhanced chemiluminescence system with the ImagePro Plus software.
7
Western Blot Analysis of Cell Markers
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Cultured cells and tissues were lysed with RIPA buffer (Beibo, China) with phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and then protein for further western blot following the protocol as describe previously [38 (link)]. The antibodies were used in present study as following: α-SMA (ab5694, Abcam), E-cadherin (ab40772, Abcam), Collagen1 (ab34710, Abcam), Fibronectin1 (FN1, ab2413, Abcam), sirt1 (ab110304, Abcam), Foxo3 (ab23683, Abcam), p-Akt (phospho Ser473, ab81283, Abcam), Akt (ab8805, Abcam), and β-actin (sc-70319, Santa Cruz).
8
Quantifying FOXO3a and FOXM1 Expression in Tumor Tissues
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IHC staining was implemented to acquire the expression of FOXO3a and FOXM1 in the
tumor tissues. 4-μM-thick paraffin-embedded sections from tumor xenografts were
prepared, blocked by 5% fetal goat serum, and respectively incubated with
anti-p-FOXO3a (ab23683, Abcam, MA, USA) and anti-FOXM1 (ab207298, Abcam, MA,
USA) antibodies. The slides were examined with an optical microscope (Olympus),
and the percentage of positive nuclei was quantified using Image J software
(NIH, Bethesda, USA).
tumor tissues. 4-μM-thick paraffin-embedded sections from tumor xenografts were
prepared, blocked by 5% fetal goat serum, and respectively incubated with
anti-p-FOXO3a (ab23683, Abcam, MA, USA) and anti-FOXM1 (ab207298, Abcam, MA,
USA) antibodies. The slides were examined with an optical microscope (Olympus),
and the percentage of positive nuclei was quantified using Image J software
(NIH, Bethesda, USA).
9
Protein Expression Analysis Protocol
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The primers used in the present experiments were showed in supplemental information . Protein extracts were obtained from cells or tissues, quantified with a bicinchoninic acid assay. The separated proteins were moved to polyvinylidene difluoride membranes and then treated with antibodies at 4 ℃ overnight 37 (link). The samples were washed and treated with the corresponding secondary antibodies for one hour 38 (link). After another washing, the membranes were treated with enhanced chemiluminescence reagents for imaging. ImageJ was used to analyze the relative expression of targeted protein bands. The primary antibodies were as follows: Foxo3 (1:1000, ab23683, Abcam), Sphk1 (1:1000, #3297, Cell Signaling Technology) and GAPDH (1:1000, ab8245, Abcam).
10
Histopathological Evaluation of Ovarian Follicles
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Following normoxic/hypoxic exposure tissue was embedded in 4% formaldehyde for further processing and analyses. Formaldehyde blocks were sectioned at 4 microns thickness. Sections were stained with H&E for follicular counts. For immunohistochemistry, sections were stained with Ki-67 (ab16667, Abcam company), with anti-Caspase 3 (ab184787, Abcam company) and anti-Foxo3A (Ab23683, Abcam company). For negative controls, the primary antibody was omitted. The slides were subjected to histopathological evaluation using Olympus microscope (BX60, serial NO. 7D04032) equipped with a camera (Olympus DP73, serial NO. OH05504) at objective magni cation of X10. The percentage of primordial, primary and secondary follicles out of total follicles per section was analyzed on H&E-stained sections. Ki-67, Caspase 3 and Foxo3A stained sections were analyzed using a semi-quantitative scoring system, as follows:
Grade 0: no signs of positive reaction.
Grade 0: no signs of positive reaction.
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