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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Mol Carcinog. 2021 Mar 8;60(5):331–341. doi: 10.1002/mc.23295

Figure 4: Inhibition of FAM129B phosphorylation affects the cellular localization of NRF2.

Figure 4:

(A) A375 cells were transfected with 80 nM BRAF siRNA twice over 72 hours prior to immunoblot analysis of BRAF, p-FAM129B, FAM129B, and NRF2. (B) NRF2/GAPDH and BRAF/GAPDH protein levels were quantified from immunoblot analyses in (A) (n=3). (C) A375 cells were transiently transfected with either 80 nM control or BRAF siRNA twice over 72 hours prior to 4 hr treatment with 10 μM U0126 or 40 nM brusatol; cells were then analyzed using indirect immunofluorescence for FAM129B (left) and β-catenin (middle). (D) Indirect immunofluorescence analysis for FAM129B (left) and KEAP1 (middle) at 4 hr post 10 μM U0126 treatment; merged panel (right) indicates colocalization of FAM129B and KEAP1. (E) A375 cells were transfected with BRAF siRNA and then subjected to indirect immunofluorescence analysis of NRF2 and nuclear counterstain Hoechst. (F) NRF2 nuclear staining was quantified as the amount of cells that had clear NRF2 nuclear localization compared to the total amount of cells (n=~50 cells across 5–6 images were quantified).