JCS010348 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Distribution of EGFP-tagged P2X₄ receptors in NRK cells. (A) Confocal images of fixed and permeabilized NRK cells stained for LAMP-1. (B) Live NRK cells with lysosomes loaded with Lysotracker by 30 minutes of incubation at 37°C. Bars, 10 μm (1 μm in enlarged panels).

• Supplemental Figure S2 -

  Fig. S2. Inhibition of endocytosis increases surface labelling of P2X₄ receptors. NRK cells co-transfected with P2X₄ (AU5) and either dynamin I (K44A) or AP180-C were surface labelled at 37°C with antibody against AU5. Cells were then fixed, permeabilized and stained using antibody against P2X₄. The AP180C and DynK44A mutants were tagged with red fluorescent protein (RFP). Bars,10 μm.

• Supplemental Figure S3 -

  Fig. S3. Role of C-terminus tyrosine-based motifs in P2X₄ targeting. (A) Surface labeling of P2X₄ (AU5) Y372F or Y378F in transfected NRK cells. Cells were surface labeled with antibody against AU5 and then fixed, permeabilized and stained with antibody against P2X₄. (B) A fusion protein containing the transmembrane and extracellular regions of CD8 fused to the C-terminus of P2X₄ (CD8 P2X₄C) was expressed in NRK cells. Cells were fixed, permeabilized and stained using antibodies against P2X4 (green) and LAMP-1 (red). The antibody against P2X₄ recognizes a C-terminal epitope within CD8 P2X₄C. Bars,10 μm.

• Supplemental Figure S4 -

  Fig. S4. No change in P2X₄ distribution following incubation with DMJ. NRK cells were incubated with DMJ (an inhibitor of Golgi α-mannosidase I) for 24 hours, fixed, permeabilized and stained with antibody against P2X₄. Bars, 10 μm.

• Supplemental Figure S5 -

  Fig. S5. ATP and MA treatments: effect on lysosomal enzyme release and cell integrity. (A) Lysosomal enzyme release in mouse peritoneal macrophages measured by assaying β-Hex release following treatment with either 1 mM ATP or 50 mM methylamine (MA) at 37°C, either with or without prior treatment with LPS. (B) Cell integrity of mouse peritoneal macrophages assayed by the release of LDH. Experiments were repeated three times and the data shown are from one representative experiment; means±s.d. (error bars), n=3.