F12 co-immunoprecipitates with kinesin light chain isoform 2.

<p>(A) SDS-PAGE and immunoblot analysis of anti-Flag immunoprecipitations. HeLa cells were transfected with plasmids expressing Flag-tagged GFP, KLC1 or KLC2 and were infected 24 h later with vF12-HA (5 PFU/cell) for 14 h. Cell lysates were prepared and immunoprecipitated with anti-Flag antibody. (i) Clarified cell lysate (Input) and immunoprecipitated samples were immunoblotted with an anti-Flag antibody. (ii) As in (i) but immunoblotted with anti-KIF5B to show equal loading of cell lysate (Input) and the ability of Flag-KLC1 and Flag-KLC2 to associate with the endogenous kinesin-1 complex (αFlag IP). (iii) As in (i) but immunoblotted with an anti-HA antibody. (B) The experiment described in (A) was repeated in HeLa cells expressing a V5 epitope-tagged A36 protein. Samples were immunoblotted with anti-V5 antibody. (C) The experiment shown in (A) (iii) was repeated in triplicate and band intensities of co-immunoprecipitated F12 were quantified using a LiCor Odyssey Infrared Imager. Numbers represent the relative integrated intensities (with local background correction) normalised to the intensity of the band in the pFlag-GFP lane of 3 independent experiments ±sd. (D) SDS-PAGE and immunoblot analysis of a reciprocal anti-HA immunoprecipitation. HeLa cells were transfected with plasmids expressing either Flag-tagged KLC1 or KLC2 and were infected 24 h later with vF12-HA or vB14-HA. HA-tagged proteins were immunoprecipitated using anti-HA antibody-coated beads. Samples were immunoblotted with (i) anti-HA, (ii) anti-Flag and (iii) anti-KIF5B (input loading control) antibodies. The positions of molecular mass markers (kDa) are shown on the left for all immunoblots.