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| Figure 2: Effect of Oct-4 Knock-down on Oct-4 mRNA and protein. [A] (i) RT-PCR analysis of Oct-4 using RNA isolated from NSCV, only cells and Oct-4 shRNA1 and 2 treated cells (ii) Relative expression of Oct-4 mRNA quantified by Q-PCR in Oct-4 shRNA1 and 2 treated, NSCV and untreated cells. [B] (i) Western blot analysis of OCT-4 normalized against GAPDH (ii) Densitometry analysis of OCT-4 protein levels in Oct-4 shRNA1 and 2 treated, NSCV and untreated cells. [C] Relative expression of Pou3f1 to rule out the possible off target effect of Oct-4 shRNAs. Pou3f1 mRNA levels did not change in Oct-4 silenced cells. Negative control did not contain Reverse transcriptase (RT) enzyme in the reaction mixture for PCR. For Western blot, replacement of primary antibody with PBS was used as a negative control. Values are the mean ± SD. n=3 different biological replicates performed on three different occasions (*p<0.05). |
