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. 2013 Apr 17;3:91. doi: 10.3389/fonc.2013.00091

Figure 1.

Figure 1

DNA-PKcs dependent phosphorylation of hnRNP A1 in MCF-10A and MCF-7. (A) DNA-PKcs siRNA knockdown. Real-time quantitative PCR assessment of DNA-PKcs mRNA relative expression from 24 to 120 h following transfection in mock (M), siRNA (S) treated cells, and the housekeeping gene Transferrin Receptor (TFRC). DNA-PKcs mRNA expression was normalized to TFRC levels in each sample and found to be maximally decreased at 24–48 h. DNA-PKcs protein levels were also assessed following siRNA transfection in mock (M), siRNA (S), or untreated (UT) cells over an identical time course; DNA-PKcs protein expression was normalized to the actin control. Optimal depletion of DNA-PKcs protein levels was observed at 72 h for both cell lines. An extended time course (to 240 h) monitored recovery of protein levels (not shown). (B) Overall phosphorylation status of hnRNP A1. 32P uptake experiments followed by immunoblotting demonstrated decreased hnRNP A1 32P signal with DNA-PKcs depletion (siRNA) and kinase inhibition (NU7026) in MCF-7, and following DNA-PKcs siRNA silencing in MCF-10A; no decrease of hnRNP A1 32P signal with DNA-PKcs kinase inhibition in MCF-10A was observed.