FIG. 2.

Bidirectional coimmunoprecipitation of galectin 9 and LMP1 from NPC and LCL cell extracts. (a) Coimmunoprecipitation of galectin 9 and TRAF3 with the anti-LMP1 antibody OT21C reacting on NPC and LCL rafts. Raft samples (60 μg of protein) were prepared from the C15 NPC tumor line (NPC) and two EBV-transformed B-cell lines (EBV1 and NAD+C15) and submitted to analytical immunoprecipitation using 5 μg of purified IgG covalently bound to 5 × 10⁷ magnetic beads. Precipitated proteins were analyzed in two parallel blots, one stained successively with anti-LMP1 (CS1-4) and anti-galectin 9 and the other stained with anti-TRAF3 antibodies. Input, 5 μg of raft-derived proteins saved prior to the immunoprecipitation step; mIgG1, control immunoprecipitation performed with mouse nonspecific IgG1; OT21C, LMP1 immunoprecipitation performed with OT21C (NPC, one-third of the eluted proteins; LCL, one-half of the eluted proteins). In the case of NAD+C15, there was a better recovery for the low-molecular-weight isoform of galectin 9. (b) Coimmunoprecipitation of galectin 9 with the anti-LMP1 OT21C and OT22CN antibodies reacting on whole-tumor extracts. Samples of C15 whole-tumor extracts (120 μg of protein) were submitted to immunoprecipitation using 5 μg of purified IgG coated on 5 × 10⁷ magnetic beads (without covalent binding). Precipitated proteins were analyzed in successive Western blots stained with anti-LMP1 (CS1-4) and anti-galectin 9 antibodies. Because precipitating IgG was not covalently bound to magnetic beads, it was recovered in the eluate and stained on the Western membrane with the anti-mouse conjugate, just below the LMP1 band. Input, crude sample of C15 whole-tumor extract containing 30 μg of protein saved prior to the immunoprecipitation step; mIgG1, control immunoprecipitation; OT21C, LMP1 immunoprecipitation performed with the OT21C antibody directed to the large C-terminal intracytoplasmic region (residues 290 to 318) (one-half of the eluted proteins); OT22CN, LMP1 immunoprecipitation performed with the OT22CN antibody directed to the short N-terminal intracytoplasmic region (residues 1 to 23) (one-half of the eluted proteins). (c) Lack of coimmunoprecipitation of galectin 9 with an anti-CD40 antibody reacting on whole-tumor extracts. Samples of C15 whole-tumor extracts (120 μg of protein) were submitted to immunoprecipitation using 5 μg of purified IgG coated on 5 × 10⁷ magnetic beads. Precipitated proteins were analyzed in parallel Western blots stained with anti-CD40 (MAb 89) and anti-galectin 9 antibodies. Protein gels prepared for CD40 detection were run in nondenaturing conditions as required for MAb 89 staining, thereby giving several bands and precluding molecular weight determination (8). Input, crude sample of C15 whole-tumor extract containing 40 μg of protein saved prior to the immunoprecipitation step; mIgG1, control immunoprecipitation; OT21C, LMP1 immunoprecipitation performed with the OT21C antibody (one-half of the eluted proteins); G28-5, CD40 immunoprecipitation performed with the G28-5 antibody (one-half of the eluted proteins). (d) Coimmunoprecipitation of LMP1 with an anti-galectin 9 antibody reacting on NPC rafts and whole-tumor extracts. C15 rafts (30 μg of protein) were submitted to immunoprecipitation using 2.5 μg of purified rabbit anti-galectin 9 Ig (loaded on 20 μl of protein A beads). Whole-tumor extracts (120 μg of protein) were treated with 3.75 μg of Ig loaded on 30 μl of beads. Precipitated proteins were analyzed in parallel Western blots stained with anti-galectin 9 (same rabbit polyclonal) and anti-LMP1 (CS1-4). Input, 10 μg of raft-derived protein or 30 μg of whole-tumor extract saved prior to the immunoprecipitation step; rIg, control immunoprecipitation performed with nonspecific rabbit Ig; gal9, galectin 9 immunoprecipitation performed with rabbit polyclonal antibodies directed to the C-terminal carbohydrate recognition domain of human galectin 9. This experiment was performed twice in duplicate. In each case, 25% and 100% of the eluted proteins were loaded in the gel for galectin 9 and LMP1 detection, respectively.