JCS034843 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Inducible Lyn and Yes are initially accumulated at the Golgi region. (A-C) HeLa cells stably expressing the tetracycline repressor (clone #3-2) were transfected with Lyn, Yes or Fyn. 12 hours after transfection, Dox was added to medium and cells were fixed at the indicated time points. Inducibly expressed proteins were detected with anti-Lyn, anti-Yes or anti-Fyn antibody. Arrows indicate the PeriN. (B) Representative results are shown and cells exhibiting the PeriN localization of expressed proteins were quantified. (C) HeLa cells transfected with Lyn or Yes were cultured for 12 hours and fixed 3 hours after induction. Cells were double stained with anti-Lyn or anti-Yes antibody (green) and anti-GM130 or anti-GalT antibody (red). Scale bars: 20 µm.

• Supplemental Figure S2 -

  Fig. S2. Precise colocalization of Yes with the Golgi pool of caveolin at the perinuclear region. COS-1 cells transfected with Yes were cultured for 1 day and double stained with anti-c-Yes (red) and anti-caveolin (green) antibodies. Arrows indicate the perinuclear region. N, nucleus. Scale bar: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Incubation at 15°C does not result in accumulation of endogenous Lyn, Yes and Fyn on ER membranes. (A-E) Dami cells were incubated for 3 hours at 15°C or 37°C and stained with anti-Lyn, anti-Yes, or anti-Fyn antibody. Cells shown in B-E were incubated at 15°C and double stained with anti-Lyn or anti-Yes antibody (green) and anti-GalT or anti-calnexin antibody (red). (F) Dami cells were incubated at 37°C and stained with anti-Fyn (green) and anti-GalT (red) antibodies. A few cells showed perinuclear staining for Fyn, whereas most of the cells showed predominant PM staining for Fyn. Arrows indicate the perinuclear region. Scale bars: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Localization of Fyn(C6S) to the Golgi region at the early phase of induction. (A,B) HeLa cells expressing the tetracycline repressor (clone #3-2) were transfected with Fyn(C6S). At 12 hours after transfection, Dox was added to the medium and fixed at the different time points. Inducibly expressed proteins were detected with anti-Fyn antibody. Arrows indicate the perinuclear region. (B) A representative experiment of Fyn(C6S) is shown. Cells exhibiting the perinuclear localization were quantified. Compare with Fig. S1A,B. (C) HeLa cells transfected with Fyn(C6S) were cultured for 12 hours and fixed at 3 hours after induction. Cells were double stained with anti-Fyn (green) and anti-GM130 (red) antibodies. Scale bars: 20 µm.

• Supplemental Figure S5 -

  Fig. S5. Comparison of pp40 in tyrosine phosphorylation between Fyn and Fyn(C6S). COS-1 cells transfected with Fyn, Fyn(C6S) or Lyn were incubated for 15 hours. Equal amounts of lysates were analyzed by western blotting with anti-phosphotyrosine (pTyr), anti-Fyn, anti-Lyn, and anti-actin antibodies. Arrows indicate tyrosine-phosphorylated proteins. Levels of the band at 40 kDa (pp40) were increased by expression of Fyn(C6S) and Lyn. However, levels of the band at 36 kDa (pp36) were comparable among expression of Fyn, Fyn(C6S) and Lyn.