Journal: Nucleic acids research
Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.
doi: 10.1093/nar/gkae370
Figure Lengend Snippet: Figure 2. A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Tw enty -f our hours af ter knoc kdo wn, cells w ere transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assa y s after mouse A-Myb o v ere xpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assa y s with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 / M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANO V A (* P < 0.05; ** P < 0.01; *** P < 0.001).
Article Snippet: For protein detection, these antiodies were used: β-actin (A5441, Sigma-Aldrich), A-MYB HPA008791, Sigma-Aldrich), B-MYB (A 301–655A, Bethyl aboratories / Fortis), CDC25C (H-6, Santa Cruz Biotechology), Cyclin B2 (A-2, Santa Cruz Biotechnology), E2F4 E3G2G rabbit mAb, 40291, Cell Signaling Technology), hisone 3 (D2B12 XP® rabbit mAb, 4620S, Cell Signaling Techology), LIN9 (A300-BL2981, Bethyl Laboratories / Fortis), IN37 (T3, custom-made at Pineda Antikörper-Service, erlin, Germany) (Müller et al., 2016), LIN54 (A303-799A, ethyl Laboratories / Fortis), Survivin (71G4B7, Cell Signaling echnology).
Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification
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