Figure 2.

Bassoon interacts with DLC1 and DLC2 in mammalian cells. (A–E) Mito-targeting assays in COS-7 cells. Cells were fixed 18 h after transfection. Mito-targeted EGFP or EGFP-DLCs are localized at mitochondria (compare A1–E1 with Fig. S1). Bsn7 shows a uniform cytoplasmic distribution when coexpressed with Mito-EGFP control construct (A2) but is corecruited to mitochondria when mito-targeted DLC1 or DLC2 are coexpressed (C2 and E2). Targeting of DLC to mitochondria does not affect the localization of mRFP (B2 and D2). The arrows in A1 indicate the normally distributed localization of mitochondria in cell protrusions. In contrast, mitochondria are clustered near the cell centers when Mito-EGFP-DLC1 and Mito-EGFP-DLC2 are expressed (C1 and E1, arrows). (F and G) Coimmunoprecipitation of the Bassoon–DLC1/2 complexes from HEK293T cells. Lysates from transfected cells with EGFP-tagged Bsn7, Bsn7^I,II,III, Pclo1, or Pclo2 and Flag-tagged DLCs were incubated with anti-GFP antibodies coupled to magnetic beads. WBs of input material (I and Input lanes) and eluates (E and Elution lanes) detected with antibodies against GFP (top), anti-Flag (F, bottom), or with DLC8 antibody (G, bottom). All expressed EGFP-tagged proteins (I and Input lanes) were successfully immunoprecipitated (E and Elution lanes, arrows). (F) DLC1 and DLC2 were coimmunoprecipitated with EGFP-Bsn7 but not with EGFP. (G) Overexpressed DLC1 and endogenous DLC were precipitated with Bsn7 but not with the DBM Bsn7^I,II,III, Pclo1 and Pclo2 constructs, and EGFP. Black lines indicate that intervening lanes have been spliced out. (F and G) Molecular mass is indicated in kilodaltons. IP, immunoprecipitation. Bar, 10 µm.