Research Paper Volume 12, Issue 7 pp 5675—5692

Figure 3. STAT5A identified as transcription factor that regulates the transcription of LINC01198. (A) Binding motif of STAT5A (TTCNNNGAA, here N denotes any nucleotide) in the promoter of LINC01198, predicted by JASPAR (http://jaspar.genereg.net/). (B) Representative binding position of STAT5A in the promoter of LINC01198 and the specific sequence of probe and its competitor probe involved in EMSA. (C) CHIP-qPCR analysis of transcriptional regulation of STAT5A on the promoter of LINC01198. Anti-STAT5A, CHIP-grade primary antibody to STAT5A; IgG, the isotype control of the primary antibody to STAT5A. The experiment was performed independently three times with triplicate in each time. Shown was the representative result. Two tailed, independent sample T-test was used to analyze the significant difference. ** P<0.01 compared with control. (D) EMSA analysis of the binding ability of STAT5A with the promoter of LINC01198. Here, competitor was the contraction of competitor probe. (E) Indirect regulation of STAT5A exerted by IL-7 (30ng/mL) over the expression of LINC01198 on mRNA level, as detected by qRT-PCR. (F) Similarly, indirect regulation of STAT5A exerted by IL-7 over the expression of LINC01198 on mRNA level in the presence of siRNA to STAT5A, as detected by qRT-PCR. Two-tailed, independent sample T-test was used to analyze the significant difference. ** P<0.01, *** P<0.001 in comparison with control group. (G) immunoblotting detection of the knock-down efficiency of siRNA to STAT5A in T87G and U-118MG glioma cells, shown were the representative figures picked out of candidates we collected. (H) STAT5A can be specifically and appreciably activated at its 694 Tyrosine in the presence of IL-7 whereas STAT3 can hardly be phosphorylated. (I) activated STAT5A was capable of translocation from cytoplasm to nucleus in the presence of IL-7, as exemplified by western-blot. Of note, PCNA used as internal loading control for nuclear protein. All the experiments related to western-blot were performed independently three times and presented were the representative ones singled out from candidates.