FAF1 interacts with VAPA and VAPB. (A) Flag-FAF1 immunoprecipitated from U2OS cells interacts with p97, VAPA and VAPB. (B) Flag-VAPA/B immunoprecipitated from U2OS cells interact with p97 and FAF1. (C) VAPB interaction with p97 is dependent on FAF1. U2OS cells were treated with the indicated siRNA oligos; luciferase (Luc) siRNA was used as a control. Flag-VAPB was immunoprecipitated and immunoblots of the immunoprecipitates (right) show that FAF1 depletion reduces the interaction with p97 whereas p97 depletion does not significantly affect the interaction with FAF1. (D) Endogenous VAPB interacts with p97 and FAF1 in mouse brain. Endogenous VAPB was immunoprecipitated from mouse brain extracts using Protein A-Sepharose (PAS) beads cross-linked to anti-VAPB antibodies. Uncoupled beads were used as a control. (E) Endogenous FAF1 interacts with VAPB in U2OS cells. The immunoprecipitation was performed using sheep anti-FAF1 antibody or sheep immunoglobulin G (IgG) as a control and PAS beads. (F) Indirect immunofluorescence of VAPB and wild-type (WT) Flag-FAF1. U2OS cells expressing Flag-FAF1 from a tetracycline-inducible promoter were grown in the presence of 200 ng/ml tetracycline for 24 hr and treated with 10 μM MG132 for 2 hr. Flag-FAF1 WT (red) co-localizes with VAPB (green) in a peri-nuclear area (enlarged window), suggesting an ER pattern. Scale bar is 10 μm. (G) VAPB levels and its interaction with Flag-FAF1 are not affected upon proteasome inhibition. Flag-FAF1 was immunoprecipitated from U2OS cells treated with 10 μM MG132 for 2 hr, 5 μM MG132 for 6 hr or left untreated (0 hr). Ubiquitinated proteins, p97 and VAPB were detected by immunoblotting in inputs (left) and immunoprecipitates (right). DAPI, 4',6-diamidino-2-phenylindole; IgG, immunoglobulin G; IP, immunoprecipitate; Luc, luciferase; WT, wild type.