sc 222 wb  (Santa Cruz Biotechnology)

Journal:

Article Title: Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

doi: 10.1073/pnas.071043198

Figure Lengend Snippet: Down-regulation of hTERT/telomerase activity is closely associated with changes in c-Myc and Mad1 expression during the differentiation of HL60 cells. HL60 cells were treated with DMSO for 48 h and analyzed for (a) hTERT mRNA expression by competitive RT-PCR; (b and c) expression of hTERT protein by immunofluorescence and Western blot; (d) telomerase activity by telomeric repeat amplification protocol assay; (e) c-Myc, Max, and Mad1 expression by Northern blot (Top), the ethidium bromide-stained gel visualizing the 18 and 28 S RNA (Middle), and Mnt expression by RT-PCR (Bottom); and (f) EMSA demonstrating DNA binding activities to the hTERT E-box in HL60 extracts. Antibodies used for supershifts are indicated at the top, and protein complexes binding to the hTERT oligo are indicated to the left. Log, logarithmically growing cells; DMSO, DMSO-treated (differentiated) cells; C, competitor for hTERT PCR product; β2-M, β2-microglobulin, internal control for the hTERT and Mnt RT-PCR.

Article Snippet: For antibody supershifts, 0.05 μg of anti-Max (sc-197), 0.2 μg of anti-Mnt (sc-769), 0.2 μg of anti-c-Myc antibody (sc-764), or 0.2 μg of anti-Mad1 (sc-222) (all from Santa Cruz Biotechnology) were added to the binding reactions.

Techniques: Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Amplification, Northern Blot, Staining, Binding Assay

Journal:

Article Title: Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

doi: 10.1073/pnas.071043198

Figure Lengend Snippet: In vivo binding of c-Myc/Max, Mad1/Max, Mnt/Max, and USF to the hTERT proximal promoter in HL60 cells. A ChIP assay was performed on logarithmically growing and DMSO-treated HL60 cells, and the precipitated chromatin was PCR-amplified with the use of specific primers. (a) (Left) Schematic presentation of E-boxes (□) in the hTERT promoter and in the E22a locus, and the location of the respective primer sequences (■) for PCR analysis. The numbers below the hTERT promoter indicate the position of the PCR primers relative to ATG. The size of the E22a PCR product is shown. (Right) In vivo identification of reciprocal E-box occupancy by c-Myc, Max, and Mad1 at the hTERT promoter in logarithmically growing (log) and differentiated (DMSO) HL60 cells. β-Galactosidase antibodies were used as controls. Input: PCRs performed on total chromatin from differentiated HL60 cells. There is an absence of c-Myc, Max, and Mad1 binding to the control E22a locus that contains E-boxes but is not transcribed. (b) Absence of USF and presence of Mnt at the hTERT promoter in vivo in undifferentiated (−) and DMSO-differentiated (+) HL60 cells. Neither Mnt nor USF was present at the control E22a locus.

Article Snippet: For antibody supershifts, 0.05 μg of anti-Max (sc-197), 0.2 μg of anti-Mnt (sc-769), 0.2 μg of anti-c-Myc antibody (sc-764), or 0.2 μg of anti-Mad1 (sc-222) (all from Santa Cruz Biotechnology) were added to the binding reactions.

Techniques: In Vivo, Binding Assay, Amplification