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Iκbα l35a5

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IκBα (L35A5) is a recombinant protein produced by Cell Signaling Technology. It is a member of the inhibitor of nuclear factor kappa-B (IκB) family, which regulate the activity of NF-κB transcription factors.

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Lab products found in correlation

Rela d14e12, by Cell Signaling Technology (2 mentions) Cd45ro uchl1, by BD (2 mentions) Cd3 hit3a, by BioLegend (2 mentions) Cd4 l200, by BD (2 mentions) Cd45ra hi100, by BD (2 mentions) Phospho s6 n7 548, by BD (2 mentions) Phospho erk1 2 20a, by BD (2 mentions) Phospho p65 k10 895, by BD (2 mentions) Phospho p38 36 p38, by BD (2 mentions) Ahr d5s6h, by Cell Signaling Technology (1 mentions) Ch 223191, by Selleck Chemicals (1 mentions) Bay 11 7085, by MedChemExpress (1 mentions) Phospho iκbα 14d4, by Cell Signaling Technology (1 mentions) α tubulin dm1a, by Cell Signaling Technology (1 mentions) Laminb1 d9v6h, by Cell Signaling Technology (1 mentions) β actin, by Beyotime (1 mentions) Gapdh, by Beyotime (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Cox 2, by Cell Signaling Technology (1 mentions) Lipopolysaccharide lps l2880, by Merck Group (1 mentions)

5 protocols using iκbα l35a5

1

Investigating HTLV-1 Activation Pathway

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CH-223191 was purchased from Selleck. L-kynurenine and BAY11-7085 were purchased from MedChemExpress. Antibodies were used as following: AHR (D5S6H; Cell Signaling Technology), RelA (D14E12; Cell Signaling Technology), IκBα (L35A5; Cell Signaling Technology), phospho-IκBα (14D4; Cell Signaling Technology), α-Tubulin (DM1A; Cell Signaling Technology), LaminB1 (D9V6H; Cell Signaling Technology), HTLV-1 Tax (1A3; Abcam), HTLV-1 gp46 (67/5.5.13.1; Abcam), HTLV-1 p24 (46/3.24.4; Abcam), HTLV-1 p19 (TP-7; Abcam), β-Actin (AF0003; Beyotime Biotechnology), GAPDH (AF0006; Beyotime Biotechnology).
Hong W., Cheng W., Zheng T., Jiang N, & Xu R. (2020). AHR is a tunable knob that controls HTLV-1 latency-reactivation switching. PLoS Pathogens, 16(7), e1008664.
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2

Ginkgo biloba Extract Inhibits Inflammation

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Ginkgo biloba L. leaf extract injection (GBE) was purchased from Youcare Pharmaceutical Group (Beijing, China, Approval No. H20070226, Supporting Information 1). Its initial concentration is 3.5 mg/ml and prepared in aqueous solvent with excipients such as sorbitol, ethanol, sodium hydroxide. Lipopolysaccharide (LPS, #L2880) were obtained from sigma, and prepared in DMEM medium at concentration of 10 mg/ml. Primary antibodies for COX-2 (#4842, 1:1000), IκB-α (L35A5) (#4814, 1:1000), p-IκB-α (14D4) (#2859, 1:1000), NF-κB p65 (D14E12) (#8242, 1:1000) and p-p65 (93H1) (#3033, 1:1000), NF-κB p50 (D4P4D) (#13586, 1:1000) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, United States). Primary antibodies for TNF-α (7B8A11) (#60291-1-Ig, 1:1000), IL-6 (#21865-1-AP, 1:1000), GAPDH (1E6D9) (#60004-1-Ig, 1:1000), β-actin (2D4H5) (#66009-1-Ig, 1:1000) and Lamin B1 (#12987-1-AP, 1:1000) were obtained from Proteintech Group Inc. (Rosemount, IL, United States). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco Laboratories.
Liu M., Peng Y., Che Y., Zhou M., Bai Y., Tang W., Huang S., Zhang B., Deng S., Wang C, & Yu Z. (2022). MiR-146b-5p/TRAF6 axis is essential for Ginkgo biloba L. extract GBE to attenuate LPS-induced neuroinflammation. Frontiers in Pharmacology, 13, 978587.
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3

Western Blot Analysis of Cellular Protein Markers

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The following antibodies were used: MCPyV Ab5 [36 (link), 57 (link)]; GFP (D5.1, Cell Signaling); vinculin (H-10, Santa Cruz); actin (D6A8, Cell Signaling); HK2 (C64G5, Cell Signaling); LDHA (EP1565Y, Abcam); MCT1 (A1512, NeoBiolab); LAT1 (5347, Cell Signaling); RelA (D14E12, Cell Signaling); IκBα (L35A5, Cell Signaling); MYC (9E10, Santa Cruz); MYCN (9405, Cell Signaling); MYCL (AF4050, R&D Systems).
Whole cell lysates were prepared using RIPA buffer (Boston BioProducts) supplemented with protease inhibitor cocktail set I (Calbiochem) and phosphatase inhibitor cocktail set I (Calbiochem). Clarified protein extracts were boiled in SDS sample buffer (Boston BioProducts), resolved by SDS-PAGE (Criterion TGX precast gels; Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), blocked and incubated with the appropriate primary antibody in TBS-T overnight at 4°C. Detection of proteins was performed with horseradish peroxidase-conjugated secondary antibodies (Rockland), developed using Clarity Western ECL substrate (Bio-Rad), and imaged with a G:BOX Chemi detection system (Syngene).
Berrios C., Padi M., Keibler M.A., Park D.E., Molla V., Cheng J., Lee S.M., Stephanopoulos G., Quackenbush J, & DeCaprio J.A. (2016). Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation. PLoS Pathogens, 12(11), e1006020.
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4

PBMC Signaling Pathway Analysis

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PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 106/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO2 incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.
Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.
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5

PBMC Signaling Pathway Analysis

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PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 106/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO2 incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.
Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.
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