Hook2 acts as a dynein–dynactin linker. (A) Domain architecture of Hook2 and its domain deletion fragments/mutants used in the study. (B) GST or GST-tagged LIC1 (389–523 aa) bound to glutathione beads were incubated with MBP-tagged Hook2 N427 (WT, Q143A, and I150A), and immunoblotted (IB) with an anti-MBP antibody for Hook2 (WT/mutants). LIC1 in the pelleted beads was detected using Ponceau S staining of the membrane. The asterisk indicates BSA protein band used for blocking glutathione beads. (C) Ratio of band intensity of pulldown to input Hook2 fragment signals in B (n = 3). (D) HEK293T cell lysates were incubated with MBP alone or MBP-tagged Hook2 N427 (WT, Q143A, and I150A) bound to amylose beads, and IB for DIC and p150^glued. The amount of recombinant Hook2 (WT/mutants) protein was analyzed by Coomassie staining. (E) Ratio of band intensity of pulldown to input Hook2 (WT/mutants) signal in D (n = 3). (F) Protein-A/G beads bound to control IgG or anti-Hook2 antibody were incubated with HEK293T lysates; the interactome IP was IB to check the presence of different dynein subunits. (G) Protein-A/G beads bound to antibodies against DIC, p150^glued, Arp1, and p50/dynamitin were incubated with HEK293T lysate; the interactome IP was IB to check the presence of Hook2. (H) Lysates from HEK293T cells treated with control or Hook2 siRNA and transfected with indicated plasmids were incubated with protein-G beads bound to antibodies against DIC and p150^glued, and IP were IB with the indicated antibodies. Arrows mark Hook2 (WT) transfected lanes. (I) Ratio of normalized band intensity (EV) of IP DIC to p150^glued and vice versa in H (n = 2). (J) Representative images of FRB-FKBP12-rapamycin dimerization assay in fixed HeLa cells. Bars, 10 µm. (K and L) Mitochondrial distribution quantified as intensity with respect to relative distance from the nucleus (n = 3; 10 cells/experiment). Data represent mean ± SD (***, P < 0.001; Student’s t test).