Prmt5

Manufactured by Abcam

Sourced in United States, United Kingdom

PRMT5 is a protein arginine N-methyltransferase that catalyzes the formation of monomethylated and symmetrical dimethylated arginine residues on target proteins. It plays a role in the regulation of gene expression and cellular processes.

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Lab products found in correlation

Gapdh, by Abcam (3 mentions) β actin, by Merck Group (3 mentions) β actin, by Abcam (3 mentions) Bca kit, by Thermo Fisher Scientific (3 mentions) Vinculin, by Merck Group (2 mentions) Image studio lite software, by LI COR (2 mentions) Peroxidase labelled anti mouse and anti rabbit igg, by Vector Laboratories (2 mentions) Srsf1, by Abcam (2 mentions) Irdye 680rd goat anti mouse igg, by LI COR (2 mentions) Irdye 800cw goat anti rabbit igg, by LI COR (2 mentions) Lamin b1, by Abcam (2 mentions) Donkey anti mouse 680rd, by LI COR (2 mentions) Odyssey clx, by LI COR (2 mentions) Image studio software, by LI COR (2 mentions) Sym10, by Merck Group (2 mentions) Nitrocellulose membrane, by Merck Group (2 mentions) Sds loading buffer, by Beyotime (2 mentions) Ecl kit, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Gapdh, by Boster Bio (1 mentions)

15 protocols using prmt5

Western Blot Protein Analysis

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The following primary and secondary antibodies were used: PRMT5 (Abcam, ab109451), Vinculin (Sigma, SAB4200080), SDMA (Cell Signalling, 13222), GAPDH (Abcam, ab181602), SRSF1 (Abcam, ab38017), Lamin B1 (Abcam, ab16048), b-Actin (Abcam, ab6276), peroxidase-labelled anti-mouse and anti-rabbit IgG (Vector Laboratories), IRDye® 800CW Goat anti-Rabbit IgG and IRDye® 680RD Goat anti-Mouse IgG (LI-COR). Western blotting quantification was performed using Image Studio Lite software (Li-COR Biosciences).

Radzisheuskaya A., Shliaha P.V., Grinev V., Lorenzini E., Kovalchuk S., Shlyueva D., Gorshkov V., Hendrickson R.C., Jensen O.N, & Helin K. (2019). PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia. Nature structural & molecular biology, 26(11), 999-1012.

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Western Blot Analysis of Protein Modifications

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Proteins were extracted using lysis buffer (125 mM Tris-HCl pH 6.8, 25% glycerol, 5% SDS) supplemented with protease inhibitor (Roche), separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto nitrocellulose membranes (HAHY00010, Millipore). The membranes were blocked in PBST containing 5% skim milk and then incubated with primary antibody. After incubation with a secondary antibody (donkey-anti-mouse or donkey-anti-rabbit, IRDye 700 or IRDye 800, respectively), signals were quantified using an Odyssey infrared imaging system (LI-COR) at 700 nm or 800 nm. The following primary antibodies were used: SDMA (Cell Signaling), PRMT5 (Abcam), HNF4α (Santa Cruz), H4R3me2s (Active Motif), and GAPDH (Boster).

Zheng B.N., Ding C.H., Chen S.J., Zhu K., Shao J., Feng J., Xu W.P., Cai L.Y., Zhu C.P., Duan W., Ding J., Zhang X., Luo C, & Xie W.F. (2019). Targeting PRMT5 Activity Inhibits the Malignancy of Hepatocellular Carcinoma by Promoting the Transcription of HNF4α. Theranostics, 9(9), 2606-2617.

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Immunohistochemical Analysis of CD11c and PRMT5

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An HRP/DAB Detection IHC Kit (Abcam, Cambridge, USA) was used for IHC staining. Briefly, primary rabbit monoclonal antibody against CD11c (Abcam) or PRMT5 (Abcam) and a primary antibody control were applied to lymph node slices or gingiva tissue slices individually and incubated overnight at 4 °C, followed by incubation with a biotinylated secondary anti-rabbit antibody (Abcam) for 10–15 min at room temperature. After 3–4 washes, the slices were incubated with streptavidin peroxidase to amplify the signal, followed by incubation with the DAB chromogen for final coloration.

Mi W., Qiao S., Zhang X., Wu D., Zhou L, & Lai H. (2021). PRMT5 inhibition modulates murine dendritic cells activation by inhibiting the metabolism switch: a new therapeutic target in periodontitis. Annals of Translational Medicine, 9(9), 755.

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Protein Expression Analysis in Lung Tissue

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Right lung lobes were homogenized and lysed in RIPA buffer (10 mM Tris pH 8.0, 5 M NaCl, 0.5 M EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) containing protease and phosphatase inhibitors (ThermoFisher Scientific). Primary antibodies: PRMT5 (Abcam ab31751, 1:1000), H4R3 (MilliporeSigma SAB4300870, 1:500), SYM10 (MilliporeSigma 07–412, 1:300), ROR-γt (Life Technologies 14–6981-82, 1µg/ml) and β-actin (Sigma-Aldrich A1978, 1:50,000). Secondary antibodies (1:20,000): donkey anti-rabbit or anti-rat 800CW and donkey-anti-mouse 680RD (Li-cor). Blots were imaged on an Odyssey-CLx and quantified with Image Studio software (Li-Cor).

Lewis B.W., Amici S.A., Kim H.Y., Shalosky E.M., Khan A.Q., Walum J., Gowdy K.M., Englert J.A., Porter N.A., Grayson M.H., Britt RD J.r, & Guerau-de-Arellano M. (2022). PRMT5 in T cells drives Th17 responses, mixed granulocytic inflammation and severe allergic airway inflammation. Journal of immunology (Baltimore, Md. : 1950), 208(7), 1525-1533.

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Cellular Fractionation and Protein Analysis

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Proteins were extracted using M-PER (ThermoScientific) for whole-cell lysate and QProteome cell compartment kit (Qiagen) for nuclear and cytoplasmic fractions. Protein was quantified using the BCA Kit (Thermo), analyzed by electrophoreses on 12% NuPAGE Novex Bis-Tris gels (Invitrogen) and transferred to Hybond ECL Nitrocellulose Membrane (GE Healthcare) according to standard procedures. Primary antibodies: PRMT5 (Abcam, Cat#12191) at 1:1000, OCT4 (Santa Cruz, Cat#sc-8628) at 1:1000, NANOG (Abcam, Cat#ab21624) at 1:300, H2A (Abcam, Cat#18255) at 1:1000, H2AR3me2s (Abcam, Cat# ab22397) at1:1000, H3 (Abcam, Cat#ab1791) at 1:1000 and β-actin (Abcam, Cat#ab8227) at 1:1000. Secondary HRP-conjugate antibodies were from Santa Cruz, all used at 1:5000 dilution. Blots were developed using ECL Western Blotting Detection Kit (GE Healthcare) according to manufacturer’s instructions. Ponseau S (Sigma) staining was performed according to standard procedures.

Gkountela S., Li Z., Chin C.J., Lee S.A, & Clark A.T. (2014). PRMT5 is required for human embryonic stem cell proliferation but not pluripotency. Stem cell reviews, 10(2), 230-239.

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Western Blot Analysis of Protein Modifications

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Cells were harvested and lysed in 1× SDS loading buffer (Beyotime), and proteins were transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% nonfat milk and then were incubated with primary antibody overnight at 4°C on a rotator. Then, the membranes were incubated with a secondary antibody, and the bands were visualized using an ECL kit (Thermo Fisher Scientific). The following primary antibodies were used: PRMT5 (#ab109451, Abcam), JAK2 (#3230T, Cell Signaling Technology), P-JAK2 (#8082T, Cell Signaling Technology), STAT1 (#9172S, Cell Signaling Technology), P-STAT1 (#7649T, Cell Signaling Technology), ACTIN (#abs830031, Absin), GAPDH (#abs830030, Absin), H3 (#ab1791, Abcam), H3R2me2s (#ab194684, Abcam), H3R8me2s (#A-3706, EpiGentek), H4R3me2s (#ab5823, Abcam) and H4 (#2935S, Cell Signaling Technology).

Jiang Y., Yuan Y., Chen M., Li S., Bai J., Zhang Y., Sun Y., Wang G., Xu H., Wang Z., Zheng Y, & Nie H. (2021). PRMT5 disruption drives antitumor immunity in cervical cancer by reprogramming T cell-mediated response and regulating PD-L1 expression. Theranostics, 11(18), 9162-9176.

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Protein Expression Analysis by Western Blot

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Cell pellets were lysed using RIPA buffer (Sigma-Aldrich, St Louis, MO, USA) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany), and phosphatase inhibitor cocktail (Roche). Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) was used to determine the protein concentration. Equal amounts of protein were loaded on to a 4–20% Tris–HCl gel (Bio-Rad, Hercules, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Antibodies PRMT5, H4R3 and GAPDH procured from abcam (Cambridge, MA, USA), Rb, AKT, pAKT, ERK, pERK, p53, PTEN and p21 obtained from (Cell Signaling, Danvers, MA, USA), p27 (R&D, Minneapolis, MN, USA) were used at the dilution of 1:000–1:2000.

Banasavadi-Siddegowda Y., Russell L., Frair E., Karkhanis V., Relation T., Yoo J., Zhang J., Sif S., Imitola J., Baiocchi R, & Kaur B. (2016). PRMT5–PTEN molecular pathway regulates senescence and self-renewal of primary glioblastoma neurosphere cells. Oncogene, 36(2), 263-274.

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Western Blot Protein Analysis

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Immunoblotting Assay for Protein Expression

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Immunoblot analyses were performed following standard procedures as described^{60 (link)}. The following antibodies for western blotting were purchased from Abcam: PRMT5 (cat. ab31751, dilution 1:1000), MSI2 (cat. ab76148, dilution 1:2000), H3R2me2 (cat. ab194684, dilution 1:1000), H4R3me2 (cat. ab5823, dilution 1:500), HEXIM1 (cat. ab25388, dilution 1:1000) and NOXA (cat. ab13654, dilution 1:500) were purchased from Abcam. P53 (cat. 2527, dilution 1:1000), c-MYC (cat. 9402, dilution 1:1000), BCL-2 (cat. 4223, dilution 1:5000), BAX (cat. 2774, dilution 1:1000), MEP50 (cat. 2823, dilution 1:1000), SmD3me2 (cat. 13222, dilution 1:1000), PARP (cat. 9542, dilution 1:1000), Cleaved caspase-3 (cat. 9661, dilution 1:1000), P21 (cat. 2947, dilution 1:5000), PUMA (cat. 12450, dilution 1:1000), MDM2 (cat. 86934, dilution 1:1000) CYCLIN B1 (cat. 12231, dilution 1:1000), CDK4 (cat. 12790, dilution 1:1000), CHK1 (cat. 2360, dilution 1:1000), RAD51 (cat. 8875, dilution 1:500), GFP (cat. 2956, dilution 1:1000) and α-TUBULIN (cat. 3873, dilution 1:10000) were purchased from Cell Signaling Technology. β-ACTIN (cat. 5316, dilution 1:10000) and anti-FLAG (cat. F3165, dilution 1:5000) were purchased from Sigma-Aldrich. SKA2 (cat. PA5-20818, dilution 1:500) was purchased from Invitrogen.

Erazo T., Evans C.M., Zakheim D., Chu K.L., Refermat A.Y., Asgari Z., Yang X., Da Silva Ferreira M., Mehta S., Russo M.V., Knezevic A., Zhang X.P., Chen Z., Fennell M., Garippa R., Seshan V., de Stanchina E., Barbash O., Batlevi C.L., Leslie C.S., Melnick A.M., Younes A, & Kharas M.G. (2022). TP53 mutations and RNA-binding protein MUSASHI-2 drive resistance to PRMT5-targeted therapy in B-cell lymphoma. Nature Communications, 13, 5676.

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Western Blotting Analysis of Cellular Proteins

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For Western blotting analysis, cells were harvested on ice and lysed as previously described¹⁶ Equal amounts of proteins (30 µg) per sample were electrophoresed and, after transferring to nitrocellulose membranes (Bio‐Rad), were incubated overnight at 4°C with the following antibodies: (Abcam, Cat #ab96231, RRID:AB_10677616), PRMT5 (Abcam, Cat #ab109451, RRID:AB_10863428), N‐cadherin (Santa Cruz Biotechnology, Cat #sc‐31030, RRID:AB_2077520), E‐cadherin (Santa Cruz Biotechnology, Cat #sc‐7870, RRID:AB_2076666), smooth muscle actin (BioGenex, Cat #MU128‐UC, RRID:AB_2335623), RB (Santa Cruz Biotechnology, Cat #sc‐50, RRID:AB_632339), phospho‐RB (Cell Signaling Technology, Cat #3590, RRID:AB_2177182), β‐actin (Sigma‐Aldrich, Cat #A5316, RRID:AB_476743) and GAPDH (Santa Cruz, Cat #sc‐2577, RRID:AB_10167668).
Membranes were washed with TBS with 0.1% Tween‐20 and incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Membranes were washed before chemiluminescence detection using Clarity ECL reagents (Bio‐Rad, Cat #1705061).

Barbarino M., Cesari D., Bottaro M., Luzzi L., Namagerdi A., Bertolino F.M., Bellan C., Proietti F., Somma P., Micheli M., de Santi M.M., Guazzo R., Mutti L., Pirtoli L., Paladini P., Indovina P, & Giordano A. (2020). PRMT5 silencing selectively affects MTAP‐deleted mesothelioma: In vitro evidence of a novel promising approach. Journal of Cellular and Molecular Medicine, 24(10), 5565-5577.

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