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. 2017 Apr 5;18(6):947–961. doi: 10.15252/embr.201643147

Figure 2. FKBP8 targeted to the OMM interacts preferentially with lipidated LC3A (LC3A‐II).

Figure 2

  • A, B
    Antibodies to endogenous LC3A/B (A), or LC3A only (B), efficiently co‐precipitated endogenous FKBP8 from SH‐SY5Y cell extracts. SH‐SY5Y cells were treated with DMSO or 10 μM CCCP for 4 h and subjected to immunoprecipitation using an LC3A/B antibody or using rabbit serum (IgG) as negative control. In another set of experiments, an LC3A‐specific antibody was used for immunoprecipitation (B). Co‐precipitated FKBP8 was detected by Western blotting using a FKBP8 antibody.
  • C
    Flag‐LC3A co‐precipitated endogenous FKBP8 from HeLa cell extracts. HeLa cells were transfected with 3×Flag‐LC3A constructs and treated with DMSO or 10 μM CCCP for 4 h and subjected to immunoprecipitation using a Flag antibody. Co‐precipitated FKBP8 was detected by Western blotting using a FKBP8 antibody.
  • D
    HeLa cells were co‐transfected with GFP, GFP‐FKBP8‐ΔTM, GFP‐FKBP8‐Mito, or GFP‐FPBP8‐ER, or GFP‐FKBP8‐WT, respectively, and Flag‐LC3A expression constructs, and co‐precipitations performed using a GFP antibody. The star indicates IgG heavy chain. The blot shows one representative experiment out of three independent experiments. The ratios of LC3A II/LC3A I are shown below the upper blot.
  • E, F
    FKBP8 overexpression in HeLa FKBP8 KO cells significantly increased the amount of lipidated Flag‐LC3A in the mitochondrial fraction. HeLa WT and a CRISPR/Cas9‐generated KO clone were transfected with 3×Flag‐LC3A constructs, or 3×Flag‐LC3A and Myc‐FKBP8 expression constructs, and treated with DMSO or CCCP (30 μM; 4 h). The amounts of Flag‐LC3A in the mitochondrial fraction (40 μg) and FKBP8 in the total fractions (30 μg) were analyzed by Western blotting. Ponceau S staining is used as loading control. Cox2 was used as mitochondrial marker. The blot in (E) is representative of two independent experiments. The graph in (F) represents the average values of quantitated Flag‐LC3A II/Flag‐LC3A I ratio of two independent experiments. Error bars show the s.d.