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. 2018 Jul 6;9(52):29892–29905. doi: 10.18632/oncotarget.25648

Figure 2. Oct-1R isoform transcription and translation.

Figure 2

(A) The full length Oct-1L and Oct-1R RNAs from Namalva (lanes 1 and 2) and Raji (lanes 3 and 4) cell lines obtained by RT-PCR and resolved on 1% agarose gel. (B) in vitro translation of Oct-1A (lane 1), Oct-1L (lane 2), and Oct-1R (lane 3) ORFs cloned in the pcDNA3.1 vector verified by Westen-blot. 1 μg of Oct-1 DNA was taken in each reaction. Western-blot was probed with anti-FLAG antibodies. (C) Western-blot of the protein extracts from the native Namalwa cells (15 μg of extract, lane 1) and the extracts from the Namalwa cells transformed with FLAG- tagged Oct-1R (Namalwa +Oct-1R) probed with antibodies against the anti-N-terminal peptide, specific for Oct-1L and Oct-1R isoforms. Two different concentrations of the Namalwa +Oct-1R cell extract (3 μg of extract, lane 2, or 15 μg of extract, lane 3) are used to better visualize the recombinant Oct-1R and to compare it with the endogenous isoform. The level of Oct-1R isoform was approximately estimated as 5% of the Oct-1L isoform level in Namalva cells (lane 1) using Amersham TM ECL TM Prime Western Blotting Detection Reagent and BIO-RAD ChemiDoc TM MP Imaging System Model. Actin expression was used as a loading control (lower panel).