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Cyclophilin b

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Cyclophilin B is a protein that functions as a peptidyl-prolyl cis-trans isomerase. It catalyzes the isomerization of proline imidic peptide bonds in oligopeptides.

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Lab products found in correlation

α tubulin, by Merck Group (2 mentions) Histone h3, by Cell Signaling Technology (2 mentions) Ripa lysis buffer, by Merck Group (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Hsp90, by BD (2 mentions) Precast gradient gel, by Bio-Rad (2 mentions) Lamin b1, by Cell Signaling Technology (2 mentions) Pan tead, by Cell Signaling Technology (2 mentions) Mouse anti v5, by Thermo Fisher Scientific (2 mentions) Ecl plus detection reagent, by GE Healthcare (2 mentions) Beclin 1, by Novus Biologicals (2 mentions) Dc sign, by BD (2 mentions) Mouse anti β actin, by Merck Group (2 mentions) β actin, by Merck Group (2 mentions) Langerin, by R&D Systems (2 mentions) Gabarap, by Cell Signaling Technology (2 mentions) Cyclophilin a, by Enzo Life Sciences (2 mentions) Wnv ns3, by R&D Systems (2 mentions) Anti ha, by Zymo Research (2 mentions)

Spelling variants of this product

Anti-Cyclophilin B (4 citations) Rabbit anti-cyclophilin B (4 citations)

12 protocols using cyclophilin b

1

Hras Protein Expression and Western Blot

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After feeder subtraction, SSCs were washed once with ice-cold TBS and lysed in lysis buffer containing PMSF (Sigma), protease inhibitor (Sigma), and phosphatase inhibitor cocktails (Sigma). Protein concentration was quantified by the BCA method. Immunoblotting was performed according to standard procedures. Denatured samples were subjected to 12% SDS/PAGE gel and transferred to PVDF membrane. The following antibodies were used: pERK (9101, Cell signaling), ERK (9102, Cell Signaling), CyclophilinB (Invitrogen), and Hras (sc-520). To obtain mouse Hras protein control for western blotting, a fragment (606 bp) of Hras (NM_008284.2) was synthesized by Integrated DNA Technologies, inserted into BamHI/EcoRI sites of pCIG [47 (link)], and overexpressed in HEK293T cells.
Yamada M., Cai W., Martin L.A., N’Tumba-Byn T, & Seandel M. (2019). Functional robustness of adult spermatogonial stem cells after induction of hyperactive Hras. PLoS Genetics, 15(5), e1008139.
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2

Western Blot Analysis of FLAG-Tagged TAPBPR

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Equal cell quantities were lysed with SDS-loading dye. Proteins were separated by gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. PVDF membranes for FLAG-tagged TAPBPR proteins were blocked with 3% BSA in tris-buffered saline supplemented with 0.2% Tween 20 (TBS-T) for 30 min, incubated with 1:2000 anti-FLAG alkaline phosphatase (Sigma-Aldrich) in 1% BSA/TBS-T, washed five times with TBS-T, and then visualized with one-step bromochloroindolyl phosphate (Thermo Fisher Scientific). Cyclophilin B (PP1B) was used as a loading control; blots were blocked with 5% nonfat milk/TBS-T, incubated with 1:2000 rabbit anti-Cyclophilin B (Invitrogen), washed five times with TBS-T, incubated with 1:10,000 goat anti-rabbit horseradish peroxidase (Jackson ImmunoResearch), washed five times with TBS-T, and visualized using Clarity Western ECL Substrate (Bio-Rad).
Sun Y., Papadaki G.F., Devlin C.A., Danon J.N., Young M.C., Winters T.J., Burslem G.M., Procko E, & Sgourakis N.G. (2023). Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes. Science Advances, 9(8), eade7151.
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3

Immunoblotting of SMC Markers

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SMCs on gels (~11,000 cells/cm2) were lysed using ice-cold Tris-Triton lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) with freshly added protease inhibitors (cOmplete Mini EDTA-free protease inhibitor tablets; Roche Applied Sciences, Penzberg, Germany). Sample protein concentrations were measured with the Pierce BCA assay (Thermo Fisher) and a BioTek ELx800 absorbance microplate reader (BioTek, Winooski, VT), normalized, boiled with Laemmli buffer, separated on 4–20% Tris–glycine polyacrylamide gels and transferred to PVDF membranes via Western blotting. SMC markers were detected with primary antibodies for calponin (clone EP798Y, 1:10000; Abcam, Cambridge, MA) and smoothelin-B (clone H-300, 1:200; Santa Cruz Biotechnology, Dallas, TX), with cyclophilin B as an internal control (1:10,000, Thermo Fisher). Bands were visualized with HRP secondary antibodies and enhanced chemiluminescence using a Syngene G:Box.
Herrick W.G., Rattan S., Nguyen T.V., Grunwald M.S., Barney C.W., Crosby A.J, & Peyton S.R. (2015). Smooth Muscle Stiffness Sensitivity is Driven by Soluble and Insoluble ECM Chemistry. Cellular and molecular bioengineering, 8(3), 333-348.
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4

Silencing of DR4 and DR5 in HCMECs

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Silencing of DR4 (TNFRSF10A: siRNA ID#s16764) and DR5 (TNFRSF10B: siRNA ID#s16756) was achieved using Ambion Silencer siRNA according to the manufacturer's recommendations. HCMECs were seeded to obtain 70% confluency in 24 h. Cells were then transfected with Lipofectamine RNAiMAX (Invitrogen) and Ambion Silencer siRNA (Life Technologies) at a final concentration of 10 μM in Opti‐MEM Reduced Serum Medium (Gibco, Life Technologies). 4 h post‐transfection, the cells were supplemented with complete media for 24 h. Following the 24 h, transfection media was removed, and cells were maintained in complete media until the treatment for the experimental endpoints. The efficiency and specificity of silencing DR4 (ThermoFischer; Hs00269492_m1) and DR5 (ThermoFischer; Hs00366272_m1) (72 h post‐transfection) was confirmed by quantitative real‐time polymerase chain reaction (qRT‐PCR) and samples were normalized to cyclophilin‐B (ThermoFischer; Hs00168719_m1). RNA was extracted utilizing the miRNeasy kit (Qiagen). cDNA was obtained using the SuperScript IV VILO (Invitrogen) reverse transcription kit according to the manufacturer's protocol.
Carey A., Parodi‐Rullan R., Vazquez‐Torres R., Canepa E, & Fossati S. (2024). Homocysteine potentiates amyloid β‐induced death receptor 4‐ and 5‐mediated cerebral endothelial cell apoptosis, blood brain barrier dysfunction and angiogenic impairment. Aging Cell, 23(5), e14106.
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5

Western Blot Analysis of Cellular Proteins

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Cells were lysed in RIPA lysis buffer (Millipore) containing protease inhibitors and phosphatase inhibitors (GenDEPOT). Proteins were resolved on 4–20% precast gradient gels (Bio-Rad) and transferred to a PVDF membrane. After blocking with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 (TBST), membranes were incubated with the primary antibody followed by the secondary antibody conjugated with horseradish peroxidase. After washing, the bands were visualized with enhanced chemiluminescence substrate (Denville). Primary antibodies used are as follows: antibodies against pan-TEAD (1:1,000, Cell Signaling Technology, #13295), FLAG (1:5,000, Sigma, #F7425), HA (1:2,000, Santa Cruz Biotechnology, #sc-7392), cyclophilin B (1:5,000, ThermoFisher Scientific, #PA1–027A), YAP (1:1,000, Cell Signaling Technology, #14074), histone H3 (1:1,000, Cell Signaling Technology, #9715), Lamin B1 (1:1,000, Cell Signaling Technology, #12586), α-tubulin (1:1,000, Sigma, #T5168), HSP90 (1:5,000, BD Biosciences, #610419), and GAPDH (1:1,000, ThermoFisher Scientific, #MA5–15738).
Kim J., Piao H.L., Kim B.J., Yao F., Han Z., Wang Y., Xiao Z., Siverly A.N., Lawhon S.E., Ton B.N., Lee H., Zhou Z., Gan B., Nakagawa S., Ellis M.J., Liang H., Hung M.C., You M.J., Sun Y, & Ma L. (2018). Long non-coding RNA MALAT1 suppresses breast cancer metastasis. Nature genetics, 50(12), 1705-1715.
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6

Cardiac mRNA Expression Profiling

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Total RNA was isolated from the whole hearts of control and cardioGRKO mice with the RNeasy Mini kit and RNase‐Free DNase kit (Qiagen) according to the manufacturer's instructions. mRNA levels were measured with a CFX96 Real‐Time System C1000 Touch Thermal Cycler (Bio‐Rad). Predesigned primer/probe sets for Nr3c1, Acta1 (skeletal muscle α‐actin [Ska]), Myh7 (β‐myosin heavy chain [βMhc]), Nppb (brain natriuretic peptide [Bnp]), Atp2a2 (sarco(endo)plasmic reticulum calcium transport ATPase 2 [Serca2]), Ryr2 (ryanodine receptor 2), Slc25a4 (solute carrier family 25 member 4 [Ant‐1, Adenine Nucleotide Translocator]), Kcnq1 (potassium voltage‐gated channel subfamily Q member 1), and ppib (cyclophilin B, peptidylprolyl isomerase B) were obtained from ThermoFisher Scientific. Values measured for each primer/probe set were normalized to Ppib.
Cruz‐Topete D., Oakley R.H., Carroll N.G., He B., Myers P.H., Xu X., Watts M.N., Trosclair K., Glasscock E., Dominic P, & Cidlowski J.A. (2019). Deletion of the Cardiomyocyte Glucocorticoid Receptor Leads to Sexually Dimorphic Changes in Cardiac Gene Expression and Progression to Heart Failure. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(15), e011012.
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7

Western Blot Analysis of Cellular Proteins

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Cells were lysed in RIPA lysis buffer (Millipore) containing protease inhibitors and phosphatase inhibitors (GenDEPOT). Proteins were resolved on 4–20% precast gradient gels (Bio-Rad) and transferred to a PVDF membrane. After blocking with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 (TBST), membranes were incubated with the primary antibody followed by the secondary antibody conjugated with horseradish peroxidase. After washing, the bands were visualized with enhanced chemiluminescence substrate (Denville). Primary antibodies used are as follows: antibodies against pan-TEAD (1:1,000, Cell Signaling Technology, #13295), FLAG (1:5,000, Sigma, #F7425), HA (1:2,000, Santa Cruz Biotechnology, #sc-7392), cyclophilin B (1:5,000, ThermoFisher Scientific, #PA1–027A), YAP (1:1,000, Cell Signaling Technology, #14074), histone H3 (1:1,000, Cell Signaling Technology, #9715), Lamin B1 (1:1,000, Cell Signaling Technology, #12586), α-tubulin (1:1,000, Sigma, #T5168), HSP90 (1:5,000, BD Biosciences, #610419), and GAPDH (1:1,000, ThermoFisher Scientific, #MA5–15738).
Kim J., Piao H.L., Kim B.J., Yao F., Han Z., Wang Y., Xiao Z., Siverly A.N., Lawhon S.E., Ton B.N., Lee H., Zhou Z., Gan B., Nakagawa S., Ellis M.J., Liang H., Hung M.C., You M.J., Sun Y, & Ma L. (2018). Long non-coding RNA MALAT1 suppresses breast cancer metastasis. Nature genetics, 50(12), 1705-1715.
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8

Western Blot and Immunofluorescence Antibody Detection

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HA-tagged constructs for western blotting were detected using a 1:5000 dilution of anti-HA-peroxidase antibody (Roche clone 3F10, #12013819001). HA-tagged constructs for indirect immunofluorescence were detected using anti-HA (Zymed, #71-5500). β-actin was also detected as a loading control using a 1:10,000 dilution of mouse anti-β-actin (Sigma, A5441). A 1:3,000 dilution of goat anti-mouse (Dako, #P0447), anti-rabbit (Thermo Scientific, #P0448) or anti-chicken (Millipore, #12-341) horseradish peroxidase-conjugated antibody was used as a secondary probe. V5 tagged constructs were probed with anti-mouse V5 (Invitrogen #R960-25). Blots were developed using the ECL Plus detection reagent (GE Healthcare, #RPN2132). Antibodies to detect viral antigens, LGTV (NS3 and NS5) (previously described in Taylor et al., 2011 (link)), WNV-NS3 (R&D Systems, #AF2907) and dsRNA antibody J2 (English& Scientific Consulting, #10010200). Autophagy and cellular markers were detected using LC3B (Nanotools, #5F10), GABARAP (Cell Signaling, #E1J4E), Beclin-1 (Novus Biologicals, # 110-53818), ATG5 (Cell Signaling, #2630), p62 (BD Transduction Laboratories, #610833), cyclophilin A (Enzo, #BML-SA296-0100), cyclophilin B (Thermo Scientific, #PA1-027A), langerin (R&D Systems, #AF2088) and DC-SIGN (BD Biosciences, #551186).
Chiramel A.I., Meyerson N.R., McNally K.L., Broeckel R.M., Montoya V.R., Méndez-Solís O., Robertson S.J., Sturdevant G.L., Lubick K.J., Nair V., Youseff B.H., Ireland R.M., Bosio C.M., Kim K., Luban J., Hirsch V.M., Taylor R.T., Bouamr F., Sawyer S.L, & Best S.M. (2019). TRIM5α Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation. Cell reports, 27(11), 3269-3283.e6.
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9

Western Blot Analysis of Protein Targets

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Western blotting was performed as described [52 (link)]. The cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (GenDepot, Houston, TX, USA). The proteins were separated by SDS-PAGE and transferred onto a PVDF membrane. The membranes were blocked in 5% non-fat milk in Tris-buffered saline-Tween 20 (TBS-T) and then incubated with the specific primary antibodies. After washed in TBS-T, the membranes were incubated with an HRP-conjugated secondary antibody. The bands were visualized by chemiluminescence. The following antibodies were used: antibodies against OTUD6A (1:1000, Proteintech, Rosemont, IL, USA), MYC (1:10,000, Proteintech, Rosemont, IL, USA), HSP90 (1:5000, Santa Cruz, Dallas, TX, USA), HA (1:1000, Santa Cruz, Dallas, TX, USA), FLAG (1:2000, Proteintech, Rosemont, IL, USA), Cyclophilin B (1:5000, Thermofisher, Waltham, MA, USA), Aurora-A (1:2000, Cell Signaling, Danvers, MA, USA), phospho-Aurora-A (Thr 288) (1:500, Abclonal, Woburn, MA, USA), PLK1 (1:1000, Abclonal, Woburn, MA, USA), and phospho-PLK1 (Thr 210) (1:1000, Cell Signaling, Danvers, MA, USA).
Kim H.J, & Kim J. (2021). OTUD6A Is an Aurora Kinase A-Specific Deubiquitinase. International Journal of Molecular Sciences, 22(4), 1936.
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10

Quantifying Cellular Signaling Pathways

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Cell Signaling Technology: CD71 (#13113S), Phospho-Histone H2A.X (#9718S), β-Actin (#4970S). Thermofisher: CD71 (#MA532500), Cyclophilin B (#PA1–027A).
Arumov A., Liyanage P.Y., Trabolsi A., Roberts E.R., Li L., Ferreira B.C., Gao Z., Ban Y., Newsam A.D., Taggart M.W., Vega F., Bilbao D., Leblanc R.M, & Schatz J.H. (2020). Optimized doxorubicin chemotherapy for diffuse Large B-Cell lymphoma exploits nanocarrier delivery to transferrin receptors. Cancer research, 81(3), 763-775.
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