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Prmt5

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PRMT5 is a recombinant protein methyltransferase enzyme that catalyzes the symmetric dimethylation of arginine residues in histone and non-histone proteins. It is an essential component of several protein complexes involved in various cellular processes.

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Lab products found in correlation

Gapdh, by Thermo Fisher Scientific (1 mentions) P21 waf1 cip1, by Thermo Fisher Scientific (1 mentions) Cyclin d1, by Thermo Fisher Scientific (1 mentions) β actin, by Fortis Life Sciences (1 mentions) Insulin, by Cell Signaling Technology (1 mentions) Bca assay, by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Ne per kit, by Thermo Fisher Scientific (1 mentions) Calnexin, by Cell Signaling Technology (1 mentions) Aldoa, by Cell Signaling Technology (1 mentions) Tsg101, by Abcam (1 mentions) α tubulin, by Thermo Fisher Scientific (1 mentions) Hif 1α, by BD (1 mentions) Imagequant las 4000 imaging system, by GE Healthcare (1 mentions) Raf 1, by Cell Signaling Technology (1 mentions) Anti h4r3me2s, by Abcam (1 mentions) Stat5b, by Santa Cruz Biotechnology (1 mentions) Eif4e, by Cell Signaling Technology (1 mentions) Symmetric di methyl arginine motif sdma, by Cell Signaling Technology (1 mentions) Ripa buffer, by Boston BioProducts (1 mentions)

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Anti-PRMT5 (5 citations)

10 protocols using prmt5

1

Western Blot Antibody Panel Analysis

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Western blotting was performed as described previously [33 (link)]. The antibodies were obtained from Cell Signaling Technology (PRMT5, p21 Waf1/Cip1, GAPDH, ATM, phospho-histone H2AX (Ser139), cyclin D1, Rb, phospho-Rb (Ser780), CDK6, PARP, cleaved caspase-7), Invitrogen (CDK4), Sigma (H4R3me2s, SYM10, SYM11), Santa Cruz (p53, β-actin), and Bethyl (HdmX/MDM4).
Che Y., Liu Y., Yao Y., Hill H.A., Li Y., Cai Q., Yan F., Jain P., Wang W., Rui L, & Wang M. (2023). Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations. Blood Cancer Journal, 13(1), 27.
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2

Quantitative Western Blot Analysis

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Cells were collected and lysed with RIPA lysis buffer, followed by centrifugation at 20,000×g for 10 min at 4 °C. Protein concentration was determined by BCA assay (Sigma-Aldrich, St. Louis, USA). Cellular proteins were separated by 4.0-20.0% SDS-PAGE (Genscript, Piscataway Township, USA) and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked by TBST with 5% fat free milk, and incubated with antibodies directed against the following proteins: Insulin (1:1000) (Cell Signaling Technology, Danvers, USA, Cat # 8138S); PRMT5 (1:1000) (Cell Signaling Technology, USA, Danvers, Cat # ab31751); and β-actin (1:10000) (Sigma-Aldrich, St. Louis, USA, Cat # A5441). Immunoreactive proteins were visualized by exposure to X-ray film. Protein bands were analyzed by image-scanning.
Ma J., He X., Cao Y., O’Dwyer K., Szigety K.M., Wu Y., Gurung B., Feng Z., Katona B.W, & Hua X. (2020). Islet-Specific Prmt5 Excision Leads to Reduced Insulin Expression and Glucose Intolerance in Mice. The Journal of endocrinology, 244(1), 41-52.
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3

Protein Fractionation and Analysis

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Whole cell lysates, SDS page, and Western blot analysis were performed as previously described (6 ). Nuclear and cytoplasmic fractions were isolated using the NE-PER kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The sDMA-recognizing antibodies are given in Fig. S2A. Other antibodies used were MTAP (catalog no.: SC-100782; Santa Cruz), actin (catalog no.: A4700; Sigma), PRMT5 (catalog no.: 2252; Cell Signaling Technology), FBP1 (catalog no.: sc-271241), FBP3 (catalog no.: sc-398466), eIF4H (catalog no.: sc-515265), PSPC1 (catalog no.: sc374181), and PABN1 (catalog no.: 66807-1-Ig; Proteintech).
Tang B., Lee H.O., Gupta S., Wang L., Kurimchak A.M., Duncan J.S, & Kruger W.D. (2022). Extracellular 5′-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins. The Journal of Biological Chemistry, 298(9), 102367.
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4

Western Blot Analysis of Cellular Proteins

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Cell lysis was performed at 4 °C using ice cold RIPA lysis buffer containing 25 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 0.1% SDS. Protein extracts were analysed by SDS-PAGE and Western blotting. ECL signals were detected as described before [76 (link)]. The following antibodies were used: α-tubulin (Thermo Fischer Scientific, Waltham, MA, USA), CD63 (Merck Millipore, Burlington, MA, USA), ALDOA, calnexin, FXR2, PKM2, PRMT5 (Cell Signaling, Danvers, MA, USA), CD81 (Biolegend, San Diego, CA, USA), HIF1α (BD Biosciences, Franklin Lakes, NJ, USA), CD9, Tsg101 (Abcam, Cambridge, UK), IPO11 (Novus Biologicals, Centennial, CO, USA).
Walbrecq G., Lecha O., Gaigneaux A., Fougeras M.R., Philippidou D., Margue C., Tetsi Nomigni M., Bernardin F., Dittmar G., Behrmann I, & Kreis S. (2020). Hypoxia-Induced Adaptations of miRNomes and Proteomes in Melanoma Cells and Their Secreted Extracellular Vesicles. Cancers, 12(3), 692.
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5

Western Blot Analysis of Protein Expression

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Proteins were extracted in RIPA buffer (Boston Bioproducts, #BP-115DG) and separated by SDS-PAGE. They were then transferred onto PVDF membranes and probed with antibodies against Symmetric Di-Methyl Arginine Motif/SDMA (Cell Signaling, #13222, MultiMab rabbit monoclonal antibody mix, 1:1000), HSP90 (BD, #610418, Clone 68, 1:10,000), PRMT5 (Cell Signaling, #2252S, 1:1000), GAPDH (Santa Cruz, #sc-365062, Clone G-9, 1:1000), SOS1 (Cell Signaling, #5890, 1:1000), STAT5B (Santa Cruz, #sc-1656, 1:200), RAF1 (Cell Signaling, #9422, 1:1000), AURKB/AIM1 (BD, #611082, Clone 6, 1:1000), SNRPB (Sigma, #HPA003482, 1:200), SNRPD3 (Sigma, #HPA001170, 1:200), SART3/TIP110 (Bethyl, #A301-521A, 1:10,000), PRP3 (MBL, #D171-3, 1:1000), SNRNP40 (MBL, #RN096PW, 1:1000), anti-H4R3me2s (Abcam, #5823, 1:500) and EIF4E (Cell Signaling, #9742, 1:1000). Proteins of interest were detected with HRP-conjugated α-Rabbit (Cell Signaling, #7074, 1:3000), Rat (Cell Signaling, #7077,1:2000) and α-Mouse (Cell Signaling, #7076, 1:2000) antibodies and visualized with the Pierce ECL Western blotting substrate (Thermo Scientific) using the ImageQuant LAS 4000 imaging system (GE) or film exposure.
Braun C.J., Stanciu M., Boutz P.L., Patterson J.C., Calligaris D., Higuchi F., Neupane R., Fenoglio S., Cahill D.P., Wakimoto H., Agar N.Y., Yaffe M.B., Sharp P.A., Hemann M.T, & Lees J.A. (2017). Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma. Cancer cell, 32(4), 411-426.e11.
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6

Western Blot Analysis of EMT Markers

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Western blot analysis was performed as previously described.36 The antibodies used in the present study were as follows: PRMT5 (1:1000), Vimentin (1:1000), Snail (1:1000), LATS2 (1:1000), p‐YAP (S127) (1:1000), E‐cadherin (1:1000) and GAPDH (1:1000) were purchased from Cell Signaling Technology. YY1 (1:300), MMP9 (1:1000) and Cyclin D1 (1:500) were from Proteintech. YAP (1:2000) was provided by Abcam. Signals were detected using Chemi Dox XRS instrument plus Image lab software (BIO‐RAD).
Wang N., Wu D., Long Q., Yan Y., Chen X., Zhao Z., Yan H., Zhang X., Xu M., Deng W, & Liu X. (2020). Dysregulated YY1/PRMT5 axis promotes the progression and metastasis of laryngeal cancer by targeting Hippo pathway. Journal of Cellular and Molecular Medicine, 25(2), 946-959.
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7

Head and Neck Cancer Cell Lines

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CAL27 cells were obtained from ATCC (Manassas, VA). UM-SCC-74B cell line was obtained from the laboratory of Dr. Thomas E. Carey at the University of Michigan.[50 (link)] The identity of both the tumor cell lines was confirmed by STR genotyping (AmpFLSTR Identifiler Kit, Applied Biosystems, Carlsband, CA). The tumor cell lines were cultured in DMEM supplemented with 10% fetal bovine serum containing 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) and 1% Non-essential amino acids. Recombinant IL-6 was obtained from PeproTech (Rocky Hill, NJ). Primary antibodies against PRMT5 and Lamin/AC were purchased from Cell Signaling (Danvers, MA) and GAPDH was purchased from Millipore (Billerica, MA).
Kumar B., Yadav A., Brown N.V., Zhao S., Cipolla M.J., Wakely P.E., Schmitt A.C., Baiocchi R.A., Teknos T.N., Old M, & Kumar P. (2017). Nuclear PRMT5, cyclin D1 and IL-6 are associated with poor outcome in oropharyngeal squamous cell carcinoma patients and is inversely associated with p16-status. Oncotarget, 8(9), 14847-14859.
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8

Western Blotting for Protein Detection

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Western blotting assay was performed as described previously [38 (link)]. The following antibodies were used: Flag (cat. no. ab49763; abcam, Cambridge, MA, USA), HA (cat. no. ab1265; abcam), Myc (cat. no. ab9106; abcam), GAPDH (cat. no. 60004–1-Ig; Proteintech, Chicago, IL, USA), NONO (cat. no. 611279; BD Bioscience, Franklin lakes, NJ, USA), PRMT1 (cat. no. 2449; Cell Signaling Technology, CST, Beverly, MA, USA), PRMT5 (cat. no. D160716; Sangon Biotech, Shanghai, China), pan-ADMA (cat. no. 13522; CST), pan-MMA (cat. no. 8015; CST). Images were captured with ChemiDocTM Imaging System (Bio-Rad, Hercules, CA, USA), and the band intensity were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
Yin X.K., Wang Y.L., Wang F., Feng W.X., Bai S.M., Zhao W.W., Feng L.L., Wei M.B., Qin C.L., Wang F., Chen Z.L., Yi H.J., Huang Y., Xie P.Y., Kim T., Wang Y.N., Hou J.W., Li C.W., Liu Q., Fan X.J., Hung M.C, & Wan X.B. (2021). PRMT1 enhances oncogenic arginine methylation of NONO in colorectal cancer. Oncogene, 40(7), 1375-1389.
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9

Western Blot Analysis of Protein Modifications

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Protein extracts were resolved through 10–15% SDS-PAGE, transferred to NC membranes, and probed with primary antibodies. Peroxidase-conjugated anti-mouse or rabbit antibody was used as secondary antibody, and the antigen–antibody reaction was visualized by enhanced chemiluminescence assay. The following commercial antibodies were used: β-actin (cat. no. 3700; Cell Signaling Technology), PRMT5 (cat. no. 79998; Cell Signaling Technology), LDHA (cat. no. 3582; Cell Signaling Technology), H3R8me2s (cat. no. ab272149; Abcam), and H4R3me2s (cat. no. ab194696; Abcam).
Shen Y., Zhao P., Dong K., Wang J., Li H., Li M., Li R., Chen S., Shen Y., Liu Z., Xie M., Shen P, & Zhang J. (2022). Tadalafil increases the antitumor activity of 5-FU through inhibiting PRMT5-mediated glycolysis and cell proliferation in colorectal cancer. Cancer & Metabolism, 10, 22.
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10

Immunohistochemical Analysis of Tumor Microarrays

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Tumor microarrays (TMA) were obtained from the MD Anderson Tissue Bank after consent and approval from the Institutional Review Board at the University of Texas MD Anderson Cancer Center and used for immunohistochemistry as described elsewhere [36 (link)]. The following antibodies were used for immunohistochemistry staining using the Dako Autostainer with appropriate positive and negative controls: PRMT5 (Cell Signaling Technology), H4R3me2s (Sigma), and Ki67 (Invitrogen). Immunohistochemistry (IHC) images were semi-quantitated for 3,3’-diaminobenzidine (DAB) intensity using ImageJ (Fiji). The correlation between the indicated protein levels was determined by the Pearson chi-square test. P values were determined by chi-square test (F), or Student’s t test using the Prism software.
Che Y., Liu Y., Yao Y., Hill H.A., Li Y., Cai Q., Yan F., Jain P., Wang W., Rui L, & Wang M. (2023). Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations. Blood Cancer Journal, 13(1), 27.
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