Ab199336

Manufactured by Abcam

Sourced in United States

Ab199336 is a lab equipment product from Abcam. It is a functional reagent, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach.

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5 protocols using ab199336

Multifaceted Antibody Utilization in Cell Signaling

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During the study, various antibodies were employed, including PELI1 (ab199336, Abcam) for Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and immunoprecipitation (IP), Cyclin E1 (11554–1-AP, Proteintech), Cyclin D1 (60186–1-Ig, Proteintech), CDK7 (#2916, CST), CDK4 (GB111602–100, Servicebio), p27 (25614–1-AP, Proteintech), E-cadherin (20874–1-AP, Proteintech), N-cadherin (22018–1-AP, Proteintech), Vimentin (10366–1-AP, Proteintech), Snail (10399–1-AP, Proteintech), Alpha Tubulin (66031–1-Ig, Proteintech), RPS3 (66046–1-Ig for WB/IHC/IF, 11990–1-AP for IP, Proteintech), Ki67 (27309–1-AP, Proteintech), PCNA (10205–2-AP, Proteintech), p53 (66031–1-Ig for WB, 10442–1-AP for IP/mIHC, Proteintech), Flag (66008–4-Ig, Proteintech), HA (66006–2-Ig, Proteintech), Myc (60003–2-Ig, Proteintech). Fluorescent secondary antibodies included mouse secondary antibody (SA00009–1, Proteintech) and rabbit secondary antibody (SA00013–4, Proteintech).
Chemical reagents employed in the experiment consisted of CHX (Aladdin, China), MG132 (Sigma, USA), four-color multiplex immunohistochemistry kit (Absin, abs50012, China), PI (Solarbio, China), DAPI (Solarbio, China), and D-Luciferin Potassium salt (PerkinElmer, USA).

Fei X., Zhu C., Liu P., Liu S., Ren L., Lu R., Hou J., Gao Y., Wang X, & Pan Y. (2024). PELI1: key players in the oncogenic characteristics of pancreatic Cancer. Journal of Experimental & Clinical Cancer Research : CR, 43, 91.

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Immunohistochemical Profiling of TonEBP and PELI1

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The paraffin-embedded human brain tissue sections underwent deparaffinization and rehydration prior to antigen retrieval. Subsequently, the brain sections were incubated with the indicated primary and secondary antibodies after the inhibition of internal peroxidase activity. A DAB substrate kit (ZLI-9018; ZSGB, Beijing, China) was employed for visualization. Lastly, the brain tissue was counterstained with hematoxylin and sealed. The immunostaining intensity was scored as follows: 0 (no immunostaining), 1 (weak immunostaining), 2 (moderate immunostaining), or 3 (strong immunostaining). The percentage of positive cells was scored as 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). Finally, the immunostaining intensity score was multiplied by the percentage-of-positive-cells score to calculate each sample’s score. The antibodies used for immunohistochemical staining were rabbit anti-TonEBP (ab3446, Abcam, Waltham, USA) and rabbit anti-PELI1 (ab199336, Abcam, Waltham, USA).

Palahati A., Luo Y., Qin L., Duan Y., Zhang M., Gan H, & Zhai X. (2024). TonEBP: A Key Transcription Factor in Microglia Following Intracerebral Hemorrhage Induced-Neuroinflammation. International Journal of Molecular Sciences, 25(3), 1438.

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Quantitative Western Blot Analysis

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The cells were treated with RIPA lysis buffer and protease inhibitors on the ice for 30 min, and then the cells were collected with centrifugation (1000 r/min, 4°C for 10 min). Subsequently, the concentration of proteins in the supernatant was measured by BCA kit purchased from Shanghai Jining Biotechnology Co., Ltd. (Shanghai, China), and then it was boiled at 100°C for 5 min and added with loading buffer (5x). The proteins were separated with SDS-PAGE and then were translated on the PVDF membranes by wet translation methods. The membranes were blocked with 5% fat-free milk for 2 hours and then were incubated with the primary antibodies including anti-PELI1 (1 : 1000, Abcam, UK, ab199336), anti-IRAK1 (1 : 1000, Abcam, UK, ab208009), and β-actin (1 : 2000, Abcam, UK, ab8226) at 4°C overnight. After that, the membranes were washed with TBST for 3 times and then incubated with the second antibodies for 2 hours. Finally, the expression of the proteins was observed and quantified by a chemiluminescence detection system.

Luo S., Wu R., Li Q, & Zhang G. (2021). MiR-301a-3p Advances IRAK1-Mediated Differentiation of Th17 Cells to Promote the Progression of Systemic Lupus Erythematosus via Targeting PELI1. Journal of Healthcare Engineering, 2021, 2982924.

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Molecular Signaling Pathway Profiling

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All common chemicals were obtained from Sigma-Aldrich (Wicklow, Ireland) unless otherwise stated. Antibodies used for Western blot and co-immunoprecipitation include rabbit anti-Bid (Abcam, ab62469, 1:1000), rabbit anti-Peli1 (Abcam ab199336, 1:1000), rabbit anti-TRAF3 (Abcam, ab76147, 1:500), mouse anti-TRAF6 (Santa Cruz, sc8409, 1:200), rabbit anti-phospho-IRF3 (Cell Signaling, 4947P, 1:500), rabbit anti-phospho-TBK1 (Cell Signaling, 5483P, 1:500), rabbit anti-phospho c-Jun (Cell Signaling, 9261S, 1:1000), rabbit anti-A20/TNFAIP3 (Cell Signaling, 5630, 1:1000), rabbit anti-LC3 (Abcam, ab51520, 1:2000), rabbit anti-FLAG (OctA-Probe D-8, Santa Cruz, sc-807, 1:500), anti- α-Tubulin (Sigma-Aldrich, T6199, 1:5000), anti-β-Actin (Sigma-Aldrich, A3853, 1:5000), and anti-GAPDH (Abcam, ab8245-100,1:5000). Bortezomib was obtained from Millennium Pharmaceuticals (Cambridge, MA, USA). Lipopolysaccharide (LPS) was obtained from (Sigma-Aldrich, L4391), and PolyI:C (31852-29-6) and Pam₃CSK4 (112208-00-1) were obtained from InvivoGen.

Kinsella S., Fichtner M., Watters O., König H.G, & Prehn J.H. (2018). Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation. Journal of Neuroinflammation, 15, 130.

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Western Blot Analysis of Cardiac Proteins

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Left ventricular myocardial tissue or cardiomyocytes were isolated and lysed with cold 1 × lysis buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and then mixed with 1 × SDS sample buffer and boiled at 95 °C for 5 min. The lysate was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked and incubated with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. The signals were detected using the ECL system (Pierce) and quantified by scanning densitometry using the Image Lab analysis system (see Supplementary Material for original western blots). The membranes were stripped with stripping buffer (P0025, Beyotime, Beijing, China) and reblotted with next primary antibody. Primary antibodies: anti-Peli1 (ab199336, Abcam, Cambridge, MA, USA), anti-HNF4α (ab41898, Abcam, Cambridge, MA, USA), anti-GAPDH (AF0006, Beyotime, Beijing, China), anti-Flag (F2555, Sigma-Aldrich, St. Louis, MO, USA), anti-Myc (AF2864, Beyotime, Beijing, China), anti-His (AF2876, Beyotime, Beijing, China), anti-HA (AF2858, Beyotime, Beijing, China).

Hou Y., Shi P., Du H., Zhu C., Tang C., Que L., Zhu G., Liu L., Chen Q., Li C., Shao G., Li Y, & Li J. (2024). HNF4α ubiquitination mediated by Peli1 impairs FAO and accelerates pressure overload-induced myocardial hypertrophy. Cell Death & Disease, 15(2), 135.

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