Journal: The Journal of Biological Chemistry

Article Title: Regulation of NRF2 by stably associated phosphoinositides and small heat shock proteins in response to stress

doi: 10.1016/j.jbc.2025.110367

Figure Lengend Snippet: HSP27 stabilizes NRF2. A , MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. B , RT–PCR analysis of NFE2L2 mRNA in MDA-MB-231 cells. NFE2L2 mRNA levels were normalized to GAPDH mRNA. The graph is shown as mean ± SD of n = 4 independent experiments. C , MDA-MB-231 cells transfected with control siRNAs or siRNAs targeting HSP27 were treated with or without 10 μM MG132 for the indicated time. NRF2 protein levels were determined by IB. D and E , HCT116 ( D ) or HS578 ( E ) cells were transiently transfected with control siRNAs or siRNAs targeting HSP27. About 68 h later, transfected cells were treated with vehicle or 100 μM tBHQ for an additional 4 h, and NRF2 expression was then analyzed by IB. The graph is shown as mean ± SD. F , A549 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27, and NRF2 expression was analyzed by IB 72 h later. The graph is shown as mean ± SD. G , 0.5 μg of recombinant His-tagged NRF2 and 9 μg KEAP1 were incubated with the indicated concentrations of HSP27. NRF2 was pulled down, and the bound KEAP1 and HSP27 were analyzed by IB. For all panels , n = 3 independent experiments unless otherwise indicated. HSP27, heat shock protein 27; IB, immunoblotting; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2–related factor 2; tBHQ, tertiary butylhydroquinone.

Article Snippet: Recombinant KEAP1 protein (#11981-H20B) was purchased from Sino Biological.

Techniques: Transfection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Recombinant, Incubation, Western Blot