Assessment of Akr1c3, Akr1b10 and γ-tubulin expression in various stably and transiently transfected cell lines using immunblotting approaches.

A) Optimization of transfection using lipofectamine 2000. Lane 1 represents untreated MCF-7_CC-12 cells, lane 2 is for identical cells transfected with an empty vector (pCMV-FLAG) in the presence of lipofectamine (1:2 ratio), while lanes 3, 4 and 5 are cells transfected with FLAG-tagged AKR1c3 expression vector (pCMV-AKR1C3-FLAG) at vector to lipofectamine ratios of 1:1, 1:1.5 and 1:2, respectively. B) A representative western blot assessing Akr1C3 protein expression in MCF-7_CC-12 cells stably transfected with an empty vector (lanes 1, 2 and 3) or pAKR1C3-FLAG (lanes 4 and 5). C) A representative western blot assessing Akr1c3 protein expression in cells transfected with a random RNA sequence (scrambled) or with an AKR1C3-specific siRNAs (KD-1 and KD-2). D) A representative western blot assessing Akr1b10 and Akr1c3 protein expression in cells transfected with a scrambled siRNA or AKR1B10-specific siRNAs (KD-3 and KD-4).