Figure 2

Co-trafficking and interaction between GAPDH and Lf in cells (a) Lf and sGAPDH signals are co-localized inside cells. (b) (i) Immunogold labeling transmission electron microscopy of purified endosomes from J774 cells demonstrates the co-localization of lactoferrin (20 nm particles indicated with arrowheads) and monoclonal anti GAPDH antibody (5 nm particles marked with arrows). (ii) In place of specific antibody isotype control antibody (5 nm particle) was used. (c) Intracellular interaction of Lf and GAPDH was demonstrated by acceptor photobleaching FRET. Bleaching of Lf-TRITC or GAPDH-TRITC (acceptor) signal is accompanied by an increase in the GAPDH-FITC or Lf-FITC (donor) signal in J774 or CHO-TRVb cells respectively (arrows). (d) FRET efficiency was calculated from 25 cells in both cases. FRET efficiency was calculated using the following formula, FRET efficienc = [Donor intensity (post-bleach)-Donor intensity (pre-bleach)]/Donor intensity (post-bleach). In control experiments the FRET donor replaced by IgG-FITC. FRET experiments were conducted using a Nikon A1R confocal microscope. (e) Interaction between internalized GAPDH and Lf was further confirmed by co-immunoprecipitation from cytosolic fraction of J774 cells. Lf along with biotinylated GAPDH (to distinguish from intracellular GAPDH) was allowed to internalize into J774 cells for 1 hr at 37 °C, cells were then washed and treated with pronase. Lf was immunoprecepitated (IP) using anti-Lf antibody coupled magnabeads. Co-immunoprecipitated biotinylated GAPDH was then detected after immunoblotting (IB) with streptavidin-HRP. Control was run in parallel wherein the cytoplasmic fraction was incubated with isotype IgG coupled magnabeads.