Fig. 3.
Retrolinkin interacts directly with BPAG1n4. (A) Yeast two-hybrid assay for BPAG1n4-retrolinkin interaction. (1) Positive control of the system. (2) Cytosolic domain of retrolinkin plus ERM2 of BPAG1n4. (3) Cytosolic domain of retrolinkin plus empty vector. (4) Cytosolic domain of retrolinkin plus p150Glued. (B) Pull-down assay for BPAG1n4-retrolinkin binding activity: BPAG1n4 protein was immobilized either on anti-BPAG1n4 (lanes 3, 4, 3′, and 4′) or sham control (lanes 5 and 5′) antibody-conjugated Protein A-Sepharose 4B. The beads were incubated with either His-retrolinkin (amino acids 31–460) (lanes 3, 5, 3′, and 5′) or His-TBCB (lanes 4 and 4′) for binding activity. Lanes 1 and 2: the total lysates of purified His-tagged proteins of His-retrolinkin (RTLN) (lane 1) and His-TBCB (lane 2). Bound, beads fraction; unbound, supernatant. The blot was immunoprobed with anti-His antibody. (C) Coimmunoprecipitation of BPAG1n4 and retrolinkin from mouse brain extracts. The anti-BPAG1n4 antibody (lane 2), but not the sham control or the beads alone (lanes 3 and 4), was able to coimmunoprecipitate retrolinkin. Lane 1: total brain lysates as detection control. (D–F) Double Immuno-EM labeling of mouse sciatic nerve sections with antiretrolinkin (15 nm) and anti-BPAG1n4 (5 nm). Arrows indicate colocalization of retrolinkin and BPAG1n4. (Scale bar: 100 nm, D–F.)