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Fig. 3. L6-Ag is ubiquitylated. Cellular distribution of ubiquitylation-deficient L6-Ag. (A,B) 293T cells were transiently transfected with the plasmids encoding wild-type L6-Ag (A) or L6K^5,86 (B) and HA-tagged ubiquitin. 48 hours later, cells were lysed in 1% Triton X-100 and the immunoprecipitation (IP) was carried out using the anti-L6-Ag mAb (lanes 2) or a negative control mAb (lanes 3). The protein lysates were used as a positive control for transfection (lanes 1). Proteins were resolved in 11% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with either anti-HA polyclonal Ab or the anti-L6-Ag mAb (L6pke). (C) MCF-7 cells were co-transfected with the plasmid encoding GFP-L6-Ag [wild type (wt), or GFP-L6K^5,86] and mRFP-CD63. Co-distribution of tagged proteins was analysed 48 hours after transfection. Shown are representative images acquired using a LSM510 microscope. (D) MCF-7 cells were transfected with the plasmid encoding L6-Ag mutants and 48 hours later the cells were processed for double-immunofluorescence staining using specific Abs as described in the legend to Fig. 2. Staining was visualised using Alexa-Fluor-488-conjugated goat anti-mouse IgG2a Ab (green, L6-Ag) and Alexa-Fluor-594-conjugated goat anti-mouse IgG1 Ab (red). Shown is the co-distribution of L6-Ag mutants and Lamp2. Scale bars: 10 µm.

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