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. 2020 Jan 9;21(3):e48902. doi: 10.15252/embr.201948902

Figure 3. Phase separation and phosphorylation of p62 are needed for full activation of Nrf2 mediated by NBR1.

Figure 3

  1. Immunofluorescence microscopy. Primary hepatocytes prepared from p62 f/f mice were infected with or without adenovirus Cre recombinase for 48 h and then additionally infected with adenovirus LacZ or NBR1. Twenty‐four hours after the infection, the hepatocytes were immunostained with anti‐Keap1, anti‐p62, and anti‐NBR1 antibodies. Each inset is a magnified image. Bars: 10 μm.
  2. Immunoprecipitation analysis. Crude extracts were prepared from primary hepatocytes as described in (A) and then immunoprecipitated with anti‐FLAG (as a negative control) or anti‐NBR1 antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three separate experiments.
  3. Primary hepatocytes prepared from p62 f/f; Alb‐Cre mice were co‐infected with adenovirus GFP or NBR1 in combination with wild‐type p62 or the mutants for 48 h. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t‐test.
  4. RT–qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± SE. *P < 0.05 and **P < 0.01 as determined by Welch's t‐test.

Source data are available online for this figure.