full length srf  (OriGene)

Journal: Cancers

Article Title: Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC

doi: 10.3390/cancers13020199

Figure Lengend Snippet: ELK1 and ELK4 are essential co-factors for SRF-mediated transcriptional regulation of MDM4 in HCC. Luciferase activity of a MDM4 promoter reporter upon siRNA-mediated knockdown of ( A ) SRF, ( B ) ELK1, and ( C ) ELK4 in HepG2 and HLE cells compared to controls. ( D ) MDM4 mRNA levels after co-transfection of an ELK1 cDNA with siNS or siSRF. Transfection efficacy was confirmed by detection of ELK1 and SRF mRNA levels. ( E ) MDM4 mRNA levels following transfection of the indicated cDNA plasmids. ELK1 S383A represents an inactive variant, which cannot be activated by phosphorylation of S383 and is thus unable to initiate target gene transcription. Transfection efficacy was confirmed by detection of ELK1 and SRF mRNA levels. Data are presented as mean ± SEM. Mann-Whitney U test: * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: siNS, scrambled, nonsense; siSRF_1/_2, siELK1_1/_2 siELK4_1/_2, siRNA 1 and 2 specifically targeting SRF, ELK1 and ELK4, respectively; ELK1, ELK1 cDNA; ELK1 S383A, ELK1 S383A cDNA; GLuc, Gaussia luciferase; SEAP, Secreted Alkaline Phosphatase; norm., normalized against control.

Article Snippet: HLE cells were transiently transfected with a pCMV6-AC-GFP vector containing either a full-length human SRF cDNA (RG208596 from OriGene Technologies, Rockville, MD, USA), or a pCGN vector containing a full-length human ELK1 cDNA, an inactive ELK1 variant (ELK1 S383A cDNA), or a pCS2plus vector containing a SRF-VP16 cDNA [ ] following the manufacturer’s protocol, using Lipofectamine 3000 (Invitrogen, Karlsruhe, Germany). pCGN-ELK-1 and pCGN-ELK-1 S383A were a gift from Ron Prywes Lab (Addgene plasmids #27156 and #27160) [ ].

Techniques: Luciferase, Activity Assay, Knockdown, Cotransfection, Transfection, Variant Assay, Phospho-proteomics, MANN-WHITNEY, Control