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Signal Transduction Protein Phosphorylation Ser / Thr Kinases PKC

Anti-PKC beta 1 + PKC beta 2 (phospho T500)抗体(ab5817)

  • Datasheet
  • SDS
Reviews (2)Q&A (6)References (3)

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Western blot - Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody (ab5817)

    Key features and details

    • Rabbit polyclonal to PKC beta 1 + PKC beta 2 (phospho T500)
    • Suitable for: WB
    • Reacts with: Human
    • Isotype: IgG

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    概述

    • 产品名称

      Anti-PKC beta 1 + PKC beta 2 (phospho T500)抗体
      参阅全部 PKC beta 1 + PKC beta 2 一抗
    • 描述

      兔多克隆抗体to PKC beta 1 + PKC beta 2 (phospho T500)
    • 宿主

      Rabbit
    • 特异性

      This antibody cross-reacts with PKC alpha [pT497] (88% homologous) and partially cross-reacts with PKC gamma [pT514] (63% homologous) and epsilon [pT566] (75% homologous), as determined by peptide competition experiments.
    • 经测试应用

      适用于: WBmore details
    • 种属反应性

      与反应: Human
      预测可用于: Mouse
    • 免疫原

      Synthetic peptide corresponding to PKC beta 1 + PKC beta 2 (phospho T500).

    • 常规说明


      Protein Kinase C beta (PKC beta) is an 80 kDa member of the conventional group (cPKCs: sensitive to diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival, transcriptional regulation and tumor promotion. PKC beta has been implicated in diabetes and carcinogenesis. PKC beta isoforms 1 & 2 are phosphorylated on three sites, threonine 500 in the activation loop, beta 1 threonine 642 (beta 2 641) in the turn loop and beta 1 serine 661 (beta 2 660) in the hydrophobic loop. Phosphorylation of PKC beta 1 & 2 on threonine 500 by PDK1 is a prerequisite for its autophosphoylation and catalytic competence.

      The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

      If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

    性能

    • 形式

      Liquid
    • 存放说明

      Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
    • 存储溶液

      pH: 7.30
      Preservative: 0.05% Sodium azide
      Constituents: PBS, 50% Glycerol, 0.1% BSA
    • Concentration information loading...
    • 纯度

      Immunogen affinity purified
    • 纯化说明

      The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PKC beta. The final product is generated by affinity chromatography using a PKC beta-derived peptide that is phosphorylated at threonine 500.
    • Primary antibody说明

      Protein Kinase C beta (PKC beta) is an 80 kDa member of the conventional group (cPKCs: sensitive to diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival, transcriptional regulation and tumor promotion. PKC beta has been implicated in diabetes and carcinogenesis. PKC beta isoforms 1 & 2 are phosphorylated on three sites, threonine 500 in the activation loop, beta 1 threonine 642 (beta 2 641) in the turn loop and beta 1 serine 661 (beta 2 660) in the hydrophobic loop. Phosphorylation of PKC beta 1 & 2 on threonine 500 by PDK1 is a prerequisite for its autophosphoylation and catalytic competence.
    • 克隆

      多克隆
    • 同种型

      IgG
    • 研究领域

      • Signal Transduction
      • Protein Phosphorylation
      • Ser / Thr Kinases
      • PKC

    相关产品

    • Compatible Secondaries

      • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
      • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Isotype control

      • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)

    应用

    The Abpromise guarantee

    Abpromise™承诺保证使用ab5817于以下的经测试应用

    “应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

    应用 Ab评论 说明
    WB (1)
    1/1000. Detects a band of approximately 80 kDa.
    说明
    WB
    1/1000. Detects a band of approximately 80 kDa.

    靶标

    • 功能

      Calcium-activated and phospholipid-dependent serine/threonine-protein kinase involved in various processes such as regulation of the B-cell receptor (BCR) signalosome, apoptosis and transcription regulation. Plays a key role in B-cell activation and function by regulating BCR-induced NF-kappa-B activation and B-cell suvival. Required for recruitment and activation of the IKK kinase to lipid rafts and mediates phosphorylation of CARD11/CARMA1 at 'Ser-559', 'Ser-644' and 'Ser-652', leading to activate the NF-kappa-B signaling. Involved in apoptosis following oxidative damage: in case of oxidative conditions, specifically phosphorylates 'Ser-36' of isoform p66Shc of SHC1, leading to mitochondrial accumulation of p66Shc, where p66Shc acts as a reactive oxygen species producer. Acts as a coactivator of androgen receptor (ANDR)-dependent transcription, by being recruited to ANDR target genes and specifically mediating phosphorylation of 'Thr-6' of histone H3 (H3T6ph), a specific tag for epigenetic transcriptional activation that prevents demethylation of histone H3 'Lys-4' (H3K4me) by LSD1/KDM1A. Also involved in triglyceride homeostasis. Serves as the receptor for phorbol esters, a class of tumor promoters.
    • 序列相似性

      Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily.
      Contains 1 AGC-kinase C-terminal domain.
      Contains 1 C2 domain.
      Contains 2 phorbol-ester/DAG-type zinc fingers.
      Contains 1 protein kinase domain.
    • 翻译后修饰

      Phosphorylation on Thr-500 within the activation loop renders it competent to autophosphorylate. Subsequent autophosphorylation of Thr-642 maintains catalytic competence, and autophosphorylation on Ser-661 appears to release the kinase into the cytosol. Autophosphorylation on other sites i.e. in the N-terminal and hinge regions have no effect on enzyme activity.
    • 细胞定位

      Cytoplasm. Nucleus. Membrane.
    • Target information above from: UniProt accession P05771 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • 数据库链接

      • Entrez Gene: 5579 Human
      • Entrez Gene: 18751 Mouse
      • Omim: 176970 Human
      • SwissProt: P05771 Human
      • SwissProt: P68404 Mouse
      • Unigene: 460355 Human
      • Unigene: 207496 Mouse
      • Unigene: 446371 Mouse
      • 别名

        • KPCB_HUMAN antibody
        • PKC beta antibody
        • PKC-B antibody
        • PKC-beta antibody
        • PKCB antibody
        • Prkcb antibody
        • PRKCB I antibody
        • PRKCB II antibody
        • PRKCB1 antibody
        • PRKCB2 antibody
        • Protein kinase C beta 1 antibody
        • Protein kinase C beta 2 antibody
        • Protein kinase C beta antibody
        • Protein kinase C beta type antibody
        • protein kinase C, beta 1 polypeptide antibody
        see all

      图片

      • Western blot - Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody (ab5817)
        Western blot - Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody (ab5817)
        Peptide Competition and Phosphatase Treatment: Lysates prepared from K562 cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5817 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to PKC beta 1 & 2  [pT500] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

        Peptide Competition and Phosphatase Treatment: Lysates prepared from K562 cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5817 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to PKC beta 1 & 2 [pT500] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

      实验方案

      • Western blot protocols

      Click here to view the general protocols

      数据表及文件

      • SDS download

      • Datasheet download

        Download

      文献 (3)

      发表研究结果有使用 ab5817?请让我们知道,以便我们可以引用本数据表中的参考文章。

      ab5817 被引用在 3 文献中.

      • Asami M  et al. Dopamine and the phosphorylated dopamine transporter are increased in the diacylglycerol kinase ?-knockout mouse brain. FEBS Lett 595:1313-1321 (2021). PubMed: 33599293
      • Molè D  et al. Protein kinase C: a putative new target for the control of human medullary thyroid carcinoma cell proliferation in vitro. Endocrinology 153:2088-98 (2012). WB ; Human . PubMed: 22374978
      • Spalding AC  et al. Inhibition of protein kinase Cbeta by enzastaurin enhances radiation cytotoxicity in pancreatic cancer. Clin Cancer Res 13:6827-33 (2007). PubMed: 18006785

      客户评价及客户问答

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      1-8 of 8 Abreviews or Q&A

      Immunocytochemistry/ Immunofluorescence abreview for Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody

      Good
      Abreviews
      Abreviews
      Application
      Immunocytochemistry/ Immunofluorescence
      Sample
      Rat Cell (Cerebellar neurons in culture)
      Specification
      Cerebellar neurons in culture
      Fixative
      Formaldehyde
      Blocking step
      Gelatin from fish as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.25
      Read More
      The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

      Abcam user community

      Verified customer

      提交于 Apr 09 2007

      Western blot abreview for Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody

      Good
      Abreviews
      Abreviews
      abreview image
      Application
      Western blot
      Sample
      Human Cell lysate - whole cell (BxPC3 cells)
      Loading amount
      20 µg
      Specification
      BxPC3 cells
      Treatment
      10% FBS
      Blocking step
      BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5%
      Read More
      The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

      Abcam user community

      Verified customer

      提交于 Oct 23 2006

      Question

      thank you for your reply. I do use protease- and phosphatase-inhibitors in my lysis buffer, so I don't think, this is the reason that the antibody is not working. Also, I use stimulated B cells in which PKCbeta should be active. Since you don't have a positive control for this product available and I do not have the cell line which you mentioned, can you tell me, if EGF-stimulated A-431 cells would be a suitable control? Thank you

      Read More

      Abcam community

      Verified customer

      Asked on Jun 29 2006

      Answer

      Further to a follow up email from our source of this antibody I have ascertained that A431 cells are apparently NOT a good choice for the detection of PKC beta. According to them the cells have been shown to express PKC alpha, delta and zeta but not PKC beta. They question the specificity of the antibody that you have been using to detect PKC beta; perhaps it is detecting generic/pan PKC? They suggest that many of the antibodies on the market demonstrate this reactivity. Jurkat or K562 cells are the best positive control cell lines according to their recommendation. There are likely to be many other cell lines in the literature. However, A431 cells are NOT suitable according to their recommendations. Additional info: K562 and Jurkats were treated with PMA, 100 nM for 10 min.; K562 had the higher expression level and better looking signal so were used those for validation purposes. In the absence of any stimulation, one should be able to detect the phosphorylation of PKCbI&II (in response to endogenous stimuli, e.g., cAMP and others). I recommend that he use Jurkat cells treated with PMA as positive control. Let me know if I can assist further. In light of this information I would like to recommend that an alternative positive control is employed. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

      Read More

      Abcam Scientific Support

      回复于 Jul 11 2006

      Question

      thank you for your reply. I do use protease- and phosphatase-inhibitors in my lysis buffer, so I don't think, this is the reason that the antibody is not working. Also, I use stimulated B cells in which PKCbeta should be active. Since you don't have a positive control for this product available and I do not have the cell line which you mentioned, can you tell me, if EGF-stimulated A-431 cells would be a suitable control? Thank you

      Read More

      Abcam community

      Verified customer

      Asked on Jun 29 2006

      Answer

      Thank you for getting back to me. I have been unable to obtain a satisfactory answer from the source of this antibody. However, I see no reason why the positive control that you have been using would not be suitable. Furthermore the approach that you are using is one I that I would recommend. I am certainly prepared to offer you a credit note against this purchase provided that the antibody was purchased within the past 90 days. If this is the case please email me details of the order including the order number and date of purchase. I look forward to hearing from you.

      Read More

      Abcam Scientific Support

      回复于 Jul 10 2006

      Question

      BATCH NUMBER 172133 ORDER NUMBER PO104494 DESCRIPTION OF THE PROBLEM I am not getting any signal at the expected size around 80kD. The only thing that comes up in a very long exposure is a weak band at around 66kD. This is not PKC beta as confirmed by blotting with control PKC beta antibody. SAMPLE Mouse B cell extract PRIMARY ANTIBODY used 1:500 in blocking buffer over night at 4 degrees wash in TBS-T DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED that is one of the things I would like to know from you ANTIBODY STORAGE CONDITIONS I stored it at -20 upon arrival, however, it was at room temperature when it arrived, because it was not shipped on dry ice. SAMPLE PREPARATION cells lyzed in buffer containing 150mM NaCl, 50mM Tris, 1mM EDTA, 10% glycerol, 2%ODG (detergent) SDS-sample-buffer containing 2-Mercaptoethanol added and sample boiled prior to gel-loading AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 9% TRANSFER AND BLOCKING CONDITIONS Tris-glycine buffer, semi-dry blotting 2h, transfer confirmed by Ponceau-stain block in 5%milk in TBS-T SECONDARY ANTIBODY anti-rabbit-HRP 1h RT(worked well on other membrane developed in parallel) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Could you please tell the specific conditions how you have used the antibody (how much protein on gel, what dilution, buffer etc). Also, is there something that I can use as apositive control to make sure that the antibody actually works? I am a bit concerned, because it arrived at room temperature. Thank you, Alina

      Read More

      Abcam community

      Verified customer

      Asked on Jun 27 2006

      Answer

      Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and I have a few comments. The approach that you are employing is largely one that I would recommend. I am however concerned that you are not detecting the phosphorylated form of PKC beta given as a result of the low level of phosphorylation of this protein in B cells. We recommend that PMA stimulated K562 cells are used to guarantee high levels of phosphorylation and good signal by western blotting. Unfortunately we do not sell this as a pre-prepared lysate. However, I would like to suggest that a stimulated cell line is incorporated into your experiments to guarantee that you have phosphorylated PKC on your blot for targeting by the antigen. I would also like to recommend that you incorporate protease inhibitors in your lysate preparation as the lack of signal may be the result of protein degradation. The western blotting conditions employed using this antibody are detailed on the antibody datasheet: Membranes were blocked with 5% BSA-TBST and incubated with the antibody diluted in 3% BSA/TBST. Should you continue to experience difficulties please get back in touch with me.

      Read More

      Abcam Scientific Support

      回复于 Jun 29 2006

      Question

      I am wondering if you have specificity information on ab5785? You note the specificity for ab5817, on the data sheet, as cross-reacting with alpha(88%), gamma(63%), and epsilon(75%). Do you have similar info. for ab5785?

      Read More

      Abcam community

      Verified customer

      Asked on Dec 08 2005

      Answer

      Thank you for your enquiry. This testing has not been performed as far as I know, but with some research I have found the following information: PKC alpha shows ~79% homology within the range of the immunogen sequence. PKC beta I shows 50% homology within the range of the immunogen sequence. PKC delta shows ~18% homology within the range of the immunogen sequence. PKC epsilon shows 50% homology within the range of the immunogen sequence. PKC gamma shows ~64% homology within the range of the immunogen sequence. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

      Read More

      Abcam Scientific Support

      回复于 Dec 12 2005

      Question

      I am interested in finding a specific antibody for activated PKC beta and I'm not sure which one would be better, ab5785 or ab5817. I will be using the antibody for Western Blot analysis and also treating with LY333,351 to inhibit PKC beta I and II. My lab is concerned with PKC activation via DAG in hyperglycemia/diabetes. If you have any further information about these antibodies I would appreciate your assistance. Thank You.

      Read More

      Abcam community

      Verified customer

      Asked on Nov 15 2005

      Answer

      Thank you for your enquiry. Are you interested in detecting PKC beta 1 or 2? Ab5785 detects PKC beta 2 phosphorylated at T641, where as ab5817 detects PKC beta 1 and 2 phosphorylated at T500. For both of these antibodies there are Western blot images on the online datasheets, and we guarantee that the antibodies will work in Western blotting with human samples. I hope this helps. If you have any additional questions, please contact us again.

      Read More

      Abcam Scientific Support

      回复于 Nov 16 2005

      Question

      what are the typical conditions for PMA(TPA) stimulation of PKC ? I have found all range of time and concentrations. For instance, what are the PMA treatment conditions that have been used in the blot you showed for anti-PKC beta1-beta2 T500 (ab5817-50 ?

      Read More

      Abcam community

      Verified customer

      Asked on Jun 16 2004

      Answer

      Thank you for your enquiry. Useful working concentration range of PMA is 1–100 nM. You may need to consider testing the half-maximal effective dose of PMA in your experimental system. Please take a look at the following website, you may find some useful information on PMA and effective concentration: http://www.promega.com/cnotes/cn001/cn001_10.pdf

      Read More

      Abcam Scientific Support

      回复于 Jun 17 2004

      Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
      For licensing inquiries, please contact partnerships@abcam.com

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