Myc clone y69

Manufactured by Abcam

Sourced in Germany, United States

Myc (clone Y69) is a recombinant rabbit monoclonal antibody that recognizes the c-Myc protein. It can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.

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MYC (Y69) (8 citations) Clone Y69 (4 citations) C-MYC (clone Y69, (4 citations) Anti-MYC (clone Y69, (4 citations)

13 protocols using myc clone y69

Immunohistochemical Profiling of Lymphoma

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Immunohistochemical stainings were performed on FFPE tissue using fully automated protocols (DAKO Autostainer Link 48). Staining protocols with antibodies to MYC (clone Y69; Abcam), BCL‐2 (clone 124; DAKO), BCL‐6 (clone PG‐B6p; DAKO), Ki‐67 (MIB‐1; DAKO), MUM‐1(clone MUM1p; DAKO), CD10 (clone 56C6; DAKO) and p53 (DO‐7; DAKO) were performed. The estimation of positive staining for CD10, BCL‐6 and MUM‐1 was based on the Hans algorithm. Cut‐off values of 70% and 40% were used for BCL2 and MYC, respectively, as previously described.16

Abdulla M., Guglielmo P., Hollander P., Åström G., Ahlström H., Enblad G, & Amini R. (2019). Prognostic impact of abdominal lymph node involvement in diffuse large B‐cell lymphoma. European Journal of Haematology, 104(3), 207-213.

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Lymphoma Immunohistochemical Profiling

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Hematoxylin and eosin (H&E)-stained slides were analyzed by pathology specialists. Histological evaluation and interpretation were done according to the WHO classification of lymphatic neoplasias [13 ,14 (link)], which covered the immunohistochemical (IHC) analysis of the intrinsic markers CD10 (clone 56C6, 1:200 dilution, Leica Biosystems, Wetzlar, Germany), BCL6 (clone GI191E/A8, ready-to-use, Roche Diagnostics, Basel Switzerland), MUM1 (clone EAU32, ready-to-use, Leica Biosystems, Wetzlar, Germany), MYC (clone Y69, 1:100 dilution, Abcam, Cambridge, UK), and BCL2 (clone 124, 1:200 dilution, Dako, Agilent Technologies Company, Santa Clara, CA, USA). Additional staining for Ki-67 (clone MIB1, 1:200 dilution, Dako, Agilent Technologies Company, Santa Clara, CA, USA) was done to determine the cell proliferation index. For DLBCL cases, the cell of origin (COO) status was assigned employing the Hans classification [15 (link)].

Mokánszki A., Bicskó R., Gergely L, & Méhes G. (2021). Cell-Free Total Nucleic Acid-Based Genotyping of Aggressive Lymphoma: Comprehensive Analysis of Gene Fusions and Nucleotide Variants by Next-Generation Sequencing. Cancers, 13(12), 3032.

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Immunohistochemistry Panel for Lymphoma

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Immunohistochemistry was performed on 4-μm FFPE sections. Antibodies used in the study were CD20 (clone L26, Abcam, cutoff: 30%), CD10 (clone 56C6, Dako, cutoff: 30%), MUM1 (clone MUM1p, Dako), Bcl6 (clone LN22, Dako, cutoff: 30%), Myc (clone Y69; Abcam, cutoff: 40%) and Bcl2 (clone 124; Dako, cutoff: 50%). Cutoff scores for each antibody were described previously [2 (link), 19 (link)].

Wu S., Zhou Y., Hua H.Y., Zhang Y., Zhu W.Y., Wang Z.Q., Li J., Gao H.Q., Wu X.H., Lu T.X, & Hua D. (2018). Inflammation marker ESR is effective in predicting outcome of diffuse large B-cell lymphoma. BMC Cancer, 18, 997.

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Protein Extraction and Western Blot Analysis

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Cells were washed with ice-cold PBS and lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5% TritonX-100, 0.5 mM EDTA, pH 8) supplemented with protease inhibitor cocktails (Sigma) and phosphatase inhibitor cocktail (Roche, Sigma). Lysates were cleared by centrifugation at 15 000 rpm for 10 min at 4°C; supernatants were removed and assayed for protein concentration using the Quick Start™ Bradford Protein Assay Kit (Bio-Rad). Lysates were denatured by addition of 5× Laemmli sample buffer (1% sodium dodecyl sulfate, 300 mM Tris-HCl pH 8, 50% glycerol, 0.025% bromophenol blue and 10% 2-mercaptoethanol) and incubation for 5 min at 95°C. At least 20 μg were subjected to SDS-PAGE and transferred to PDVF membranes (Bio-Rad), following the manufacturers’ instructions. The following antibodies were used: RAS (3965, CST), MYC (clone Y69, Abcam), AKT (9272 CST), DKC1 (H-300, Santa Cruz Biotechnology), ATRX (H-300, Santa Cruz Biotechnology), ATRX N-terminal (D1N2E clone, CST), p21 (EPR362, Abcam), p53 (DO-1, Santa Cruz Biotechnology), Phospho-p53 Ser46 (2521 CST), HIPK2 (5091, CST) and β-actin (clone A-15, Sigma-Aldrich).

Beneventi G., Munita R., Cao Thi Ngoc P., Madej M., Cieśla M., Muthukumar S., Krogh N., Nielsen H., Swaminathan V, & Bellodi C. (2021). The small Cajal body-specific RNA 15 (SCARNA15) directs p53 and redox homeostasis via selective splicing in cancer cells. NAR Cancer, 3(3), zcab026.

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Immunohistochemical Analysis of Lymphoma

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IHC (Figure 6) was performed on 4 μm sections with formalin-fixed paraffin-embedded (FFPE) specimens. Antibodies applied in the study including CD10 (clone 56C6; Dako, cut-off: 30%), MYC (clone Y69; Abcam, cut-off: 40%), BCL2 (clone 124; Dako, cut-off: 50%), BCL6 (clone LN22; Dako, cut-off: 30%), and MUM1 (clone MUM1p; Dako, cut-off: 30%). The COO was classified according to Hans algorithm [32 (link)].

Lu T.X., Fan L., Wang L., Wu J.Z., Miao K.R., Liang J.H., Gong Q.X., Wang Z., Young K.H., Xu W., Zhang Z.H, & Li J.Y. (2015). MYC or BCL2 copy number aberration is a strong predictor of outcome in patients with diffuse large B-cell lymphoma. Oncotarget, 6(21), 18374-18388.

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Immunohistochemical Profiling of Lymphoma Subtypes

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Antibodies applied in the study, according to the manufacturer’s instructions, included CD5 (clone EP2952, Abcam, cut-off: 30%), CD10 (clone 56C6, Dako, cut-off: 30%), CD30 (clone CON6D/B5, Abcam, cut-off: 30%), Ki-67 (clone Mib-1, Dako), Myc (clone Y69, Abcam, cut-off: 40%), Bcl2 (clone 124, Dako, cut-off: 50%), Bcl6 (clone LN22, Dako, cut-off: 30%), MUM1 (clone MUM1p, Dako, cut-off: 30%), FOXP1 (clone JC12, Abcam, cut-off: 60%), GCET1 (clone RAM341; Abcam, cut-off: 60%) and LMO2 (clone 1A9-1, Santa Cruz, cut-off: 30%). The cell of origin (COO) was classified according to Hans, Choi, Tally and Visco-Young algorithms. The specific cut-off of each antibody used in different algorithms was described previously21 (link)22 (link)23 (link)24 (link).

Lu T.X., Liang J.H., Miao Y., Fan L., Wang L., Qu X.Y., Cao L., Gong Q.X., Wang Z., Zhang Z.H., Xu W, & Li J.Y. (2015). Epstein-Barr virus positive diffuse large B-cell lymphoma predict poor outcome, regardless of the age. Scientific Reports, 5, 12168.

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Immunohistochemical Profiling of Lymphoma

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IHC was performed on 4-μm FFPE sections. The antibodies used were CD10 (clone 56C6, Dako), CD20 (clone L26, Abcam), Bcl6 (clone LN22, Dako), and MUM1 (clone MUM1p, Dako), Myc (clone Y69; Abcam, cut-off: 40%) and Bcl2 (clone 124; Dako, cut-off: 50%) (Figure S1). The cut-off scores for each antibody were described previously9 (link)19 (link). Cases positive for both CD10 and MUM1were classified as DP group. Cases negative for CD10, Bcl6 and MUM1 were defined as TN group. Cases positive for both Myc and Bcl2 or Bcl6 were defined as double expression lymphoma (DEL)19 (link).

Lu T.X., Miao Y., Wu J.Z., Gong Q.X., Liang J.H., Wang Z., Wang L., Fan L., Hua D., Chen Y.Y., Xu W., Zhang Z.H, & Li J.Y. (2016). The distinct clinical features and prognosis of the CD10+MUM1+ and CD10−Bcl6−MUM1− diffuse large B-cell lymphoma. Scientific Reports, 6, 20465.

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Biomarker Expression in ENKL

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We measured the expression of 3 biomarkers, CD30, pSTAT3 (Tyr705-phosphorylated STAT3), and MYC, in ENKTLs (n = 71). Standard, indirect IHC was performed with antibodies specific for CD30 (clone 1G12, ready to use, Nichirei; Tokyo, Japan), pSTAT3 (clone D3A7, dilution 1:400; Cell Signaling Technology, Danvers, MA), and MYC (clone Y69, dilution 1:200, Abcam; Cambridge, UK). Membranous reactivity for CD30 and nuclear reactivity for pSTAT3 and MYC, at any intensity, were scored as positive on the single-cell level, then the sample was scored as the percentage of overall neoplastic cells in a sample with 10% increments. Expression was scored as high if positivity was ≥30% for CD30, ≥30% for pSTAT3, and ≥40% for MYC, as reported previously.²¹ (link)^,²² (link)

Oishi N., Satou A., Miyaoka M., Kawashima I., Segawa T., Miyake K., Mochizuki K., Kirito K., Feldman A.L., Nakamura N, & Kondo T. (2022). Genetic and immunohistochemical profiling of NK/T-cell lymphomas reveals prognostically relevant BCOR-MYC association. Blood Advances, 7(1), 178-189.

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Immunohistochemical Profiling of Lymphoma

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Immunohistochemistry was performed on 4-μm formalin-fixed paraffin-embedded sections. The antibodies used in the study were for CD20 (clone L26, Abcam, cutoff: 30%), CD10 (clone 56C6, Dako, cutoff: 30%), Bcl6 (clone LN22, Dako, cutoff: 30%), MUM1 (clone MUM1p, Dako, cutoff: 30%), and Myc (clone Y69; Abcam, cutoff: 40%). The cutoff values for each antibody have been previously described [7 (link)].

Lu T.X., Wu S., Cai D.Y., Hong T.T., Zhang Y., Gao H.Q., Hua H.Y, & Wu X.H. (2019). Prognostic significance of serum aspartic transaminase in diffuse large B-cell lymphoma. BMC Cancer, 19, 553.

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Molecular Profiling of Diffuse Large B-Cell Lymphoma

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Evaluation of genetic alterations and protein expression has been described previously (11 (link), 14 (link), 17 (link), 30 (link)–32 (link)). Formalin-fixed paraffin-embedded (FFPE) biopsies were organized prior to R-CHOP treatment and tissue microarray was constructed for study. IHC analysis was performed for the following markers with corresponding antibodies: MYC (clone Y69, Epitomics), BCL-2 (clone 124, DAKO), p53 (clone DO-7, DAKO), and Ki-67 (clone MIB-1, DAKO). The cut-off values were 40% for MYC, 50% for BCL-2, 30% for p53 (average of the two cutoffs in previous studies; refs. 14 (link), 17 (link)), and 70% for Ki-67 as previously determined.
FISH analysis was performed on FFPE sections to detect MYC rearrangement using a Vysis LSI MYC dual-color break-apart rearrangement probe (Abbott Molecular) following the manufacturer’s instructions. Images were captured and reviewed using Cytovision software (Applied Imaging). At least 200 tumor cell nuclei were scored and a cutoff of more than 5% of positive cells was used to determine the presence of MYC rearrangement.
TP53 mutations were detected using the AmpliChip p53 Research Test (Roche Molecular Systems), which is a microarray-based assay that detects mutations in exons 2–11 (14 (link)).

Deng M., Xu-Monette Z.Y., Pham L.V., Wang X., Tzankov A., Fang X., Zhu F., Visco C., Bhagat G., Dybkaer K., Chiu A., Tam W., Zu Y., Hsi E.D., You H., Huh J., Ponzoni M., Ferreri A.J., Møller M.B., Parsons B.M., Hagemeister F., van Krieken J.H., Piris M.A., Winter J.N., Li Y., Xu B., Liu P, & Young K.H. (2020). Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents. Molecular cancer research : MCR, 19(2), 249-260.

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