For Immunofluorescence, 6 μm frozen sections of aortic roots were fixed and permeabilized with cold-acetone for 5 min and blocked in PBS containing 3% BSA for 1 hour at room temperature. Sections were stained with rat anti-CD31 (BD Pharmingen, 550274, 1:30), rabbit anti-p16 (1:100, ab51243, Abcam), rabbit anti-p21 (1:400, 2947, Cell Signaling), rabbit anti-γH2AX (2577, 1:800, Cell Signaling), cleaved caspase3 (1:400, 9661, Cell Signaling), Importin-α3 (1:100, ab84706, Abcam) and rabbit anti-MEKK3 (Cell Signaling) in PBS at 4 oC overnight. After three washes with PBS, the sections were further incubated with Alexa Flour 488-conjugated donkey anti-Rat IgG (Invitrogen, A21208, 1:300) and Alexa Flour 555-conjugated goat anti Rabbit IgG (Invitrogen, 1:300) in PBS for 1 hour at room temperature. Nuclei were stained for 5min at room temperature in PBS containing DAPI (Cell signaling, 4083, 0.5μg/ml). Coverslips were mounted with ProLong Gold antifade reagent (Invitrogen). Images were acquired on an upright Carl Zeiss LSM 510 confocal. The data were calculated from at least 40 cells for each group with 6–10 mice. For each mouse, 2–5 plaques were selected and used for quantification. Tunnel protocol was performed as described by manufacturer’s protocol (Millipore, ApopTaq Peroxidase Detection kit).
Ab51243
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab51243 is a recombinant monoclonal antibody that detects the Neurofibromin 1 (NF1) protein. The core function of this product is to serve as a reliable detection tool for the NF1 protein in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab109520, by Abcam (9 mentions)
Ab109199, by Abcam (8 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Bca assay kit, by Thermo Fisher Scientific (3 mentions)
Ab16663, by Abcam (2 mentions)
Ab32389, by Abcam (2 mentions)
L7543, by Merck Group (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Ab125011, by Abcam (2 mentions)
Ab109201, by Abcam (2 mentions)
Ab52903, by Abcam (2 mentions)
Ab68155, by Abcam (2 mentions)
Ab188224, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ab131442, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
44 protocols using ab51243
1
Immunofluorescence Analysis of Aortic Plaque
2
Western Blot Analysis of Cell Cycle Regulators
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Total proteins of tissues and cultured cells were extracted by using RIPA buffer containing of 1% protease inhibitors (Sigma-Aldrich). The concentrations of soluble protein were measured by bicinchoninic acid (BCA) method (Beyotime, Nantong, China). Equal quality of total proteins of every group were separated with 10% SDS-PAGE (Beyotime, Nantong, China), transferred to PVDF, and blocked in 5% milk for 1 hr at RT. Membranes were incubated overnight at 4°C with the primary antibody against p16 (Abcam, ab51243), p21 (Abcam, ab109199), Cyclin E1 (Abcam, ab71535), Cyclin D1 (Abcam, ab16663), CDK4 (Abcam, ab137675), and GAPDH (Beyotime, Nantong, China). The secondary antibody against rabbit (Beyotime, Nantong, China) and mouse (Beyotime, Nantong, China) was incubated at RT for 1 hr. At last, ECL detection kit (Millipore, USA) was used to detect the protein bands.
3
Immunohistochemical Evaluation of Transplanted Tumors
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The transplanted tumors were surgically removed until the tumor volume reached 100 mm3, and then fixed in 10% formalin. Then, the tumors derived from the donor’s pelvic lymph node and the transplanted tumors were subjected to routine hematoxylin and eosin (HE) staining and staining for other markers, including P16 (1:50, ab51243, Abcam, Cambridge, UK), P53 (1:2000, ab32389, Abcam), ER (1:800, 21244-1-AP, Proteintech), PR (1:200, 25871-1-AP, Proteintech, Chicago, IL, USA), PAX8 (1:200, 10336-1-AP, Proteintech), and WT1 (1:500, ab89901, Abcam). Microscopic slides were reviewed by a senior gynecology-dedicated pathologist (Prof. Yang).
Similarly, immunohistochemistry (IHC) was conducted to assess the expression of Ki67 (1:200, AF0198, Affinity, San Francisco, CA, USA), CyclinD1 (1:250, ab134175, Abcam), Hes1 (1:200, ab108937, Abcam), N-cadherin (1:200, #13116, Cell Signaling Technology, Danvers, MA, USA), vimentin (1:200, #5741, Cell Signaling Technology) and CSF3 (1:200, 17185-1-AP, Proteintech) in the tumors extracted from mice. The kidney and liver were also removed for HE staining to evaluate the toxicity of viscera.
Finally, the expression of CSF3 in the corresponding lymph nodes samples from 10 ovarian cancer patients was evaluated by IHC. The scoring criteria for staining intensity were described in our previous research [33 (link)].
Similarly, immunohistochemistry (IHC) was conducted to assess the expression of Ki67 (1:200, AF0198, Affinity, San Francisco, CA, USA), CyclinD1 (1:250, ab134175, Abcam), Hes1 (1:200, ab108937, Abcam), N-cadherin (1:200, #13116, Cell Signaling Technology, Danvers, MA, USA), vimentin (1:200, #5741, Cell Signaling Technology) and CSF3 (1:200, 17185-1-AP, Proteintech) in the tumors extracted from mice. The kidney and liver were also removed for HE staining to evaluate the toxicity of viscera.
Finally, the expression of CSF3 in the corresponding lymph nodes samples from 10 ovarian cancer patients was evaluated by IHC. The scoring criteria for staining intensity were described in our previous research [33 (link)].
4
Autophagy and Senescence Markers Analysis
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At 0, 24 and 48 hours, NPCs were collected and lysed in lysis buffer (Beyotime) on ice using a western and IP cell lysis kit. Then, the protein extracts were collected by centrifugation at 15 000 g for 15 minutes at 4°C. Protein concentrations of cell lysates were tested using an enhanced BCA protein assay kit (Beyotime). After protein transfer, the membranes were blocked with nonfat milk and then incubated overnight at 4°C with rat antibodies against LC3B (L7543, 1:1000, Sigma), p62 (sc‐48402, 1:500, Santa Cruz, USA), p16 (ab51243, 1:1000, Abcam), p21 (ab218311, 1:1000, Abcam), p53 (sc‐126, 1:500, Santa Cruz), PARKIN (ab77924, 1:200, Abcam), PINK1 (ab23707, 1:500, Abcam), COX IV (ab202554, 1:1000, Abcam) and β‐actin (8H10D10, 1:1000, CST). After three washes, the membranes were incubated using peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Finally, the proteins were visualized using the enhanced chemiluminescence method following the manufacturer's instructions (Amersham Biosciences).
5
Immunoblotting for GAPDH, ZKSCAN3, LC3-II/I, p62, p16, and Lamin B1
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The primary antibody against glyceraldehyde-phosphate dehydrogenase (GAPDH) was obtained from absin (#Ab5830030, Shanghai, China). The primary antibody against ZKSCAN3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibody against LC3-II/I was obtained from Sigma (L7543, Saint Louis, MI, USA). The primary antibody against p62 was purchased from Abcam (Ab56416, Cambridge, UK). The primary antibody against p16 was obtained from Abcam (Ab51243, Cambridge, UK). The primary antibody against lamin B1 was purchased from Abcam (Ab16048, Cambridge, UK).
6
Western Blot Analysis of Myogenic and Exosomal Markers
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Cells or EVs were sonicated into the lysis buffer supplemented with phosphatase and protease inhibitors (KGP2100, Keygen Biotech, China). Proteins were transferred onto PVDF membranes. Then the PVDF membranes were blocked by 5% milk (P0216-1500 g, Beyotime, China) and incubated with primary antibodies against GAPDH (1:10000, HRP-60004, proteintech, USA), P16 (1:1000, ab51243, abcam, UK), P53 (1:2000, A10610, ABclonal, China), P21 (1:2000, A1483, ABclonal, China), myogenic differentiation antigen (MyoD) (1:100, ab203383, abcam, UK), myogenin (MyoG) (1:200, ab124800, abcam, UK), myosin heavy chain (MyHC) (1:1000, ab91506, abcam, UK), CD9 (1:1000, ab263019, abcam, UK), CD81 (1:1000, ab109201, abcam, UK), TGS101 (1:1000, ab125011, abcam, UK), and Calnexin (1:1000, ab133615, abcam, UK) at 4 °C overnight. Membranes were then incubated with goat anti-rabbit IgG(H + L) HRP (70-GAR0072, MultiSciences, China) at 37 °C for 1 h. Subsequently, the immune complexes were visualized using a tanon™ high-sig ECL western blotting substrate (180-5001, Tanon, China) and automatic digital gel/chemiluminescence image analysis system (4600SF, Tanon, China).
7
Western Blot Protein Analysis Protocol
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We followed our previously described protocol [31 (link)]. Briefly, frozen tissue samples were homogenised in ice-cold lysis buffer and protein concentrations determined using a BCA protein assay kit (Sigma, Poole, UK). Lysates were mixed with 3 x SDS PAGE sample buffer, boiled for 5 min and allowed to cool to room temperature. Equal amounts of protein (30–50 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, using 7.5–12.5% polyacrylamide resolving gels, and transferred onto nitrocellulose membrane (Invitrogen, Paisley, UK), and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 °C in 5% milk diluted in Tris-buffered saline (TBS) and 0.1% Tween 20 and incubated with the following primary antibodies overnight at 4 °C: anti-p21 (Cell Signaling; #2947), anti-p16 (Abcam; ab51243), or anti-cGAMP (Abcam; ab48508). After washing and incubating with secondary antibodies, immunoreactive proteins were visualized by the ECL plus chemiluminescence system following the manufacturer's instructions (Amersham Biosciences, Bucks., UK). Protein bands were quantified using Image J software (National Institutes of Health, http://rsb.info.nih.gov/ij/ ). Protein loading was normalized against Poncaeu S staining [35 (link)]. The values are expressed as a percentage of the control lysate (100%) for each experiment.
8
Protein Expression Analysis of Kidney Tissues
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The total protein of mouse kidney tissues and HK-2 cells were extracted by RIPA lysis buffer (P0013B; Beyotime, Shanghai, China). The proteins were electrophoresed in a 10%–12% SDS-PAGE gel and transferred to a Nitrocellulose Transfer Membrane (66485; Pall Corporation). After blocking the membrane was incubated with primary antibodies at 4°C overnight. The antibodies used in this study included rabbit monoclonal antibody against p16 (ab51243; Abcam), cyclin D1 (ab16663; Abcam), Smad3 (9523; Cell Signaling Technology), p-Smad3 (ab52903; Abcam) and mouse monoclonal antibody FLAG M2 (F1804; Sigma-Aldrich), β-actin (SC-69879; Santa Cruz). Then, the membranes were incubated with rabbit IgG (H&L) antibody DyLight 800 conjugated (611-145-002; Rockland) or mouse IgG (H&L) antibody DyLight 800 conjugated (610-145-002; Rockland). The expression levels of protein were visualized using the Odyssey Infrared Imaging System (San Diego, CA) and quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
9
Protein Expression Analysis of Lung Tissue
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Frozen right lung tissues were homogenized with RIPA buffer containing protease inhibitor. Aliquots of tissue lysates were used for protein analysis and then separated on 12% SDS–PAGE. Separate proteins were transferred onto the Immobilon-P transfer membranes 0.45 mm (Millipore). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST). After blocking for 3 h, the membrane was incubated with primary antibodies (1:500 dilutions) in blocking buffer for 24h at 4°C. Primary antibodies against SOD2 (ab68155), SIRT3 (ab86671), p16 (ab51243) and p21 (ab109199) were purchased from Abcam. After washing with TBST for 3 times (10 min each), the membranes were incubated with secondary anti-rabbit antibody (1:10,000 dilutions in 5% skim milk) linked to HRP for 1 h at room temperature, and then the membranes were developed using ECL method (Millipore, Billerica, U.S.A). β-actin was used as a loading control. ImageJ densitometry software (Version 1.41; National Institutes of Health, Bethesda, MD) was used for the band quantification of Western blots.
10
Immunohistochemical Analysis of p21 and p16
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The lung tissues were dehydrated and embedded in paraffin. For histological examination, 4 μm-thick sections on slides were treated with 1.4% H2O2-methanol for 30 min to block endogenous peroxidase. Then, nonspecific binding was blocked with 1.5% normal saline, and the slides were incubated with rabbit anti-p21 (1:200; #ab188224; Abcam, Cambridge, UK) and rabbit anti-p16 (1:100; #ab51243; Abcam) antibodies. The next day, the sections were incubated with ABC kit reagents (Vector Laboratories, Burlingame, CA, USA). The color reaction was developed by staining with a liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA). After immunohistochemical staining, the slides were counterstained with Harris’s hematoxylin for 1 min.
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