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Cyclophilin b

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Cyclophilin B is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. It catalyzes the interconversion of cis and trans isomers of peptide bonds with proline residues, which can be a rate-limiting step in protein folding.

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Lab products found in correlation

β actin, by Cell Signaling Technology (5 mentions) Bca protein assay, by Thermo Fisher Scientific (4 mentions) Vinculin, by Merck Group (2 mentions) Sc 59623, by Abcam (2 mentions) Phospho acc, by Cell Signaling Technology (2 mentions) β actin, by Abcam (2 mentions) Ab92513, by Abcam (2 mentions) Ab52615, by Abcam (2 mentions) Mper lysis, by Thermo Fisher Scientific (2 mentions) Ecl prime, by Cytiva (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Hsp70, by Abcam (1 mentions) Olig2, by Merck Group (1 mentions) C myc y69, by Abcam (1 mentions) Gfap g a 5, by Merck Group (1 mentions) Ki67 sp6, by Abcam (1 mentions) N myc ncm 2 100, by Abcam (1 mentions) Cdkn2a p16ink4a, by Abcam (1 mentions) β tubulin, by Cell Signaling Technology (1 mentions) Lamin b1, by Abcam (1 mentions)

12 protocols using cyclophilin b

1

Antibody Characterization for Immunostaining

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Antibodies for immunostaining and western blotting were used as per the manufacturer’s recommendations. The following antibodies were used within this study: β-tubulin (Cell Signalling Technology, Cat # 2146 (1:1000)), Cleaved Caspase-3 [Asp175] (Abcam, Cat # ab49899 (WB 1:500, IHC 1:1500)), CDKN2A/p19ARF (Abcam, Cat # ab80 (1:1000)), cMYC [Y69] (Abcam, Cat # ab32072 (WB 1:1000, IHC 1:500)), Cyclophilin B (Cell Signalling Technology, Cat # 43603 (1:1000)), GFAP [GA5] (Millipore, Cat # MAB3402 (1:1000)), GFP (Abcam, Cat # ab13970 (1:1000)), HSF1 (Abcam, Cat # ab61382 (1:1000)), HSP70 (Abcam, Cat # ab2787 (1:1000)), HSP90 [EPR16621] (Abcam, Cat # ab203085 (1:1000)), Ki67 [SP6] (Abcam, Cat # ab16667 (1:2000)), n-MYC [NCM II 100] (Abcam, Cat # ab16898 (1:250)), NPR3 (Abcam, Cat # ab97389 (1:250)), Olig-2 (Millipore, Cat # AB9610 (1:1000)), OTX2 (R&D Systems, Cat # AF1979 (1:500)), p21 (Abcam, Cat # ab109199 (1:1000)), Synaptophysin [SY38] (Millipore, Cat # MAB5258 (1:500)), TUJ1 (Covance, Cat # MMS-435P (1:500)), β-actin (Santa Cruz Biotechnology, Cat # sc-47778 (1:1000)), CDKN2A/p16INK4A (Abcam, Cat # ab211542 (1:1000)), anti-mouse IgG secondary HRP (VWR, Cat # NXA931 (1:1000)), anti-rabbit IgG secondary HRP (GE Healthcare, Cat # NA934 (1:1000)), and Lamin B1 (Abcam, Cat # ab16048 (1:1000)).
Mainwaring O.J., Weishaupt H., Zhao M., Rosén G., Borgenvik A., Breinschmid L., Verbaan A.D., Richardson S., Thompson D., Clifford S.C., Hill R.M., Annusver K., Sundström A., Holmberg K.O., Kasper M., Hutter S, & Swartling F.J. (2023). ARF suppression by MYC but not MYCN confers increased malignancy of aggressive pediatric brain tumors. Nature Communications, 14, 1221.
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2

Quantitative Western Blot Analysis

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Protein lysates were prepared in either RIPA or CHAPS buffer and quantified using the BCA Protein Assay (Thermo Scientific). Protein was separated on 4%–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against O-GlcNAcylation (Santa Cruz, sc-59623, dilution 1:200), β-actin (Abcam, ab8227, 1:5,000), cyclophilin B (clone EPR12703(B), ab178397, 1:5,000), CPS1 (ab3682, 1:2,000), GFPT1 (ab125069, 1:2,000), GFPT2 (ab190966, 1:2,000), phospho-ACC (Cell Signaling, #11818, 1:1,000), LKB1 (Cell Signaling, #3050, 1:1,000), BiP (Cell Signaling, #3177, 1:1,000), AMPK (Cell Signaling, #2603, 1:1,000), OGT (Sigma, O6264–200UL, 1:2,000), Vinculin (Sigma, V9131, 1:1,000).
Kim J., Lee H.M., Cai F., Ko B., Yang C., Lieu E.L., Muhammad N., Rhyne S., Li K., Haloul M., Gu W., Faubert B., Kaushik A.K., Cai L., Kasiri S., Marriam U., Nham K., Girard L., Wang H., Sun X., Kim J., Minna J.D., Unsal-Kacmaz K, & DeBerardinis R.J. (2020). The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer. Nature metabolism, 2(12), 1401-1412.
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3

Western Blot Analysis of HSPC Proteins

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Western blot analysis was performed in whole bone marrow cell and sort-purified HSPC lysates. Antibodies used were RIPK3 (ProSci), MLKL (3H1; EMD Millipore), caspase 8 (R & D systems), RIPK1 (BD Transduction Labs) actin (BD Transduction Labs), and cyclophilin B (Cell Signaling Technology). For analysis of MLKL in HSPCs, protein samples were concentrated using the Chemicon Protein-Concentrate kit (EMD Millipore).
Smith J.N., Zhang Y., Li J.J., McCabe A., Jo H.J., Maloney J, & MacNamara K.C. (2018). Type I IFNs drive hematopoietic stem and progenitor cell collapse via impaired proliferation and increased RIPK1-dependent cell death during shock-like ehrlichial infection. PLoS Pathogens, 14(8), e1007234.
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4

Protein Expression Analysis by Western Blot

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Boiled whole cell lysates were run on a precast Tris-acetate (Thermo Fisher Scientific) or Mini-PROTEAN TGX protein gel (Bio-Rad) and then transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated overnight at 4 °C with primary antibodies against Cas9 (1:1,000; Active Motif 61577), Pten (1:1,000; Cell Signaling Technology 9188), ERG (1:1,000; Abcam ab92513), p53 (1:1,000; Cell Signaling Technology 2524), Apc (1:1,000; Millipore MABC202), AKT1 (1:2,000, Cell Signaling Technology 4060L), AKT2 (1:1,000, Cell Signaling Technology 5239S), pPRAS40-Thr246 (1:1,000, Cell Signaling Technology 2997S), Actin-HRP (horseradish peroxidase) (1:10,000; Abcam ab49900), Cyclophilin B (1:10000; Cell Signaling Technology 43603S) and Vinculin (1:10,000; Cell Signaling Technology 13901). Signal was visualized with secondary HRP-conjugated antibodies and chemiluminescent detection.
Feng W., Cao Z., Lim P.X., Zhao H., Luo H., Mao N., Lee Y.S., Rivera A.A., Choi D., Wu C., Han T., Romero R., de Stanchina E., Carver B.S., Wang Q., Jasin M, & Sawyers C.L. (2021). Rapid interrogation of cancer cell of origin through CRISPR editing. Proceedings of the National Academy of Sciences of the United States of America, 118(32), e2110344118.
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5

Exosome Protein Isolation and Analysis

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Following incubation with exosomes for 24 h, cells and isolated exosomes were collected, washed with DPBS and lysed in radio-immunoprecipitation assay (RIPA) buffer with Halt protease and phosphatase inhibitors (all from Thermo Scientific). Protein concentration was determined using the Pierce BCA Protein Assay (Thermo Scientific). Equivalent amounts of protein lysates were loaded on to 4–20% Mini-PROTEAN® TGX gels (Bio-Rad) and transferred to EMD Millipore Immobilon™-FL PVDF (Fisher Scientific). Primary antibodies CD63 (HPA010088, Sigma), β-tubulin (T5201, Sigma), cyclophilin-B (43603S, Cell Signaling) and β -actin (3700S Cell Signaling) were used for detection. Secondary IRDye antibodies (LI-COR) were used together with the Odyssey® Fc Imaging System (LI-COR).
Shu S.L., Yang Y., Allen C.L., Maguire O., Minderman H., Sen A., Ciesielski M.J., Collins K.A., Bush P.J., Singh P., Wang X., Morgan M., Qu J., Bankert R.B., Whiteside T.L., Wu Y, & Ernstoff M.S. (2018). Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment. Scientific Reports, 8, 12905.
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6

Quantifying STAT1 Signaling Dynamics

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1x106 PBMCs/ml were incubated in 24 well- plates with 50 ng/ml of recombinant human IFN-γ (PeproTech) for 0 (at rest), 30, 120 and 180 min in complete RPMI medium. To ensure equal loading, protein concentrations were assessed via Pierce™ BCA Protein Assay Kit (Thermo Scientific). Lysates were denatured in 1x LDS buffer (Invitrogen) then boiled for 10 mins at 70°C. Lysates were then run on NuPAGE Novex 4-12% gels (Invitrogen) with Spectra Multicolour Broad Range Protein Ladder (Invitrogen). Electrophoresis was run a 350A for 1 hour to transfer protein to a PDVF membrane. Membranes were then blocked with 5% non-fat milk in 1x Tris buffered saline with 0.1% Tween20 (Sigma) (TBST) for 1 hour at room temperature. Primary antibodies used were B-actin, Phospho-STAT1, STAT1, pJAK1, STAT3, pSTAT3, Cyclophilin B, NFKB1, pAKT, AKT SOCS1, FAK, and JAK2 (all Cell Signaling). Membranes were incubated overnight with primary antibodies, then washed 3x 10mins with TBST, before being incubated with a rabbit HRP-conjugated secondary antibody for 1 hours at room temperature. Membranes were washed a further 3x 10mins with TBST then developed with chemiluminescence ECL or Femto (Thermo Fisher).
Körholz J., Gabrielyan A., Sowerby J.M., Boschann F., Chen L.S., Paul D., Brandt D., Kleymann J., Kolditz M., Toepfner N., Knöfler R., Jacobsen E.M., Wolf C., Conrad K., Röber N., Lee-Kirsch M.A., Smith K.G., Mundlos S., Berner R., Dalpke A.H., Schuetz C, & Rae W. (2021). One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency. Frontiers in Immunology, 12, 680334.
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7

Quantification of cGAS Protein Levels

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Cell pellets from GFP-cGasKI/KI and control BMDMs were lysed in 2× Laemmli buffer and incubated at 95°C for 5 min. Proteins were separated on a 12% denaturing acrylamide gel and subsequently transferred onto a nitrocellulose membrane (Amersham Hybond-ECL, GE Healthcare). Membrane was blocked using 1× RotiBlock (Carl Roth) for 1 h and incubated overnight at 4°C with the primary antibodies against cGAS (1:1,000, #31659; Cell Signaling Technology), β-actin (1:10,000, #4970; Cell Signaling Technology), and Cyclophilin B (1:20,000, #43603; Cell Signaling Technology) diluted in 1× RotiBlock. Following washing with TBS/0.1% Tween, the membrane was incubated for 1 h at room temperature with peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000, #7074; Cell Signaling Technology) and washed again. The Amersham ECL Prime Western blotting Detection Reagent (GE Healthcare) was used for protein visualization using the Fusion FX (Vilber Lourmat) and Fusion FX7 Advanced imaging software. Signals were densitometrically analyzed using ImageJ software (NIH).
Schumann T., Ramon S.C., Schubert N., Mayo M.A., Hega M., Maser K.I., Ada S.R., Sydow L., Hajikazemi M., Badstübner M., Müller P., Ge Y., Shakeri F., Buness A., Rupf B., Lienenklaus S., Utess B., Muhandes L., Haase M., Rupp L., Schmitz M., Gramberg T., Manel N., Hartmann G., Zillinger T., Kato H., Bauer S., Gerbaulet A., Paeschke K., Roers A, & Behrendt R. (2022). Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner. The Journal of Experimental Medicine, 220(1), e20220829.
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8

Quantitative Western Blot Analysis

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Protein lysates were prepared in either RIPA or CHAPS buffer and quantified using the BCA Protein Assay (Thermo Scientific). Protein was separated on 4%–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against O-GlcNAcylation (Santa Cruz, sc-59623, dilution 1:200), β-actin (Abcam, ab8227, 1:5,000), cyclophilin B (clone EPR12703(B), ab178397, 1:5,000), CPS1 (ab3682, 1:2,000), GFPT1 (ab125069, 1:2,000), GFPT2 (ab190966, 1:2,000), phospho-ACC (Cell Signaling, #11818, 1:1,000), LKB1 (Cell Signaling, #3050, 1:1,000), BiP (Cell Signaling, #3177, 1:1,000), AMPK (Cell Signaling, #2603, 1:1,000), OGT (Sigma, O6264–200UL, 1:2,000), Vinculin (Sigma, V9131, 1:1,000).
Kim J., Lee H.M., Cai F., Ko B., Yang C., Lieu E.L., Muhammad N., Rhyne S., Li K., Haloul M., Gu W., Faubert B., Kaushik A.K., Cai L., Kasiri S., Marriam U., Nham K., Girard L., Wang H., Sun X., Kim J., Minna J.D., Unsal-Kacmaz K, & DeBerardinis R.J. (2020). The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer. Nature metabolism, 2(12), 1401-1412.
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9

Protein Extraction and Western Blotting

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Cells were plated and treated with PP121 or DMSO for 3 h, rinsed with PBS and lysed using CelLytic M Cell lysis reagent (Sigma-Aldrich; Merck KGaA). Protein concentrations were subsequently determined using Bradford reagent. Proteins (30 µg) were separated via SDS-PAGE in 8% polyacrylamide gels and transferred to PVDF membranes. Membranes were incubated with primary antibodies at 1:500 against phosphorylated (p)-Akt (product no. 4060L), Akt (product no. 4691), p-S6 ribosomal protein (p-RPS6; product no. 4858S), S6 ribosomal protein (product no. 2317), and cyclophilin B (product no. 43603; all from Cell Signaling Technology, Inc.) overnight at 4˚C. Following primary incubation, membranes were incubated with an HRP-conjugated secondary antibody (product no. 7074S; Cell Signaling Technology, Inc.) at 1:1,000 for 1 h at room temperature, and visualized using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Inc.), and a UVP BioSpectrum imaging system (Analytik Jena AG).
, & Quick Q.A. (2023). Efficacy of PP121 in primary and metastatic non‑small cell lung cancers. Biomedical Reports, 18(4), 29.
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10

Immunoblotting Analysis of Cell Signaling

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Immunoblotting was performed by standard method as previously described (24 (link)). The primary antibodies used in this study included p53 (#2524), p-p53 (s15) (#9284, #9286), PARP (#9532), cleaved caspase-3 (#9664), Bak (#12105), PUMA (#4976), HA tag (#5017), cyclophilin B (#43603) and GAPDH (#5174) from Cell signaling, and p21 (ahz0422) from Invitrogen. Each experiment was repeated for at least four times, and representative images are present.
Xiang X., Zhu J., Zhang G., Ma Z., Livingston M.J, & Dong Z. (2021). Proximal Tubule p53 in Cold Storage/Transplantation-Associated Kidney Injury and Renal Graft Dysfunction. Frontiers in Medicine, 8, 746346.
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