JCS030338 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The transferrin receptor does not cluster and colocalize with GM1. (A) DRG cells were labeled with 500 nM BODIPY-GM1 and then incubated with Alexa-Fluor-546-conjugated antibody against transferrin receptor (TfR). DRG cells were analyzed by confocal laser microscopy 5 minutes after the addition of 5 μg/ml of laminin-1. Arrowheads indicate clustering of BODIPY-GM1. Graphs show the fluorescence intensity, which was measured using Leica LCS software, in arbitrary units versus the length of indicated lines 1, 2 and 3 in three clustering examples shown in the upper fluorescence image panels 5 minutes after laminin-1 addition. Laminin-1 induced focal aggregation of BODIPY-GM1 in the membrane but not of control TfR. Green, BODIPY-GM1; red, TfR. The line numbers correspond to the numbers of the graphs. Scale bars, 5 μm. (B) PC12 cells were treated with 100 ng/ml NGF and 5 μg/ml laminin-1. Cells were lysed in 1% Triton X-100 and were fractionated on discontinuous sucrose gradients. Ten fractions were collected from top to bottom of the gradient (1-10). Each fraction was subjected to immunoblotting using the indicated antibodies. Flotillin 1, a raft marker, was detected in fractions 2 and 3.

• Supplemental Figure S2 -

  Fig. S2. Clustering and colocalization of BODIPY-GM1 and TrkA in living DRG cells. DRG cells were labeled with 500 nM BODIPY-GM1 and then incubated with Alexa-Fluor-546-conjugated anti-TrkA antibody. DRG cells were analyzed by confocal laser microscopy at 5 minutes after the addition of 5 μg/ml of laminin-1. Arrowheads indicate clustering of BODIPY-GM1 (Green) and TrkA (red). The graphs in the bottom panel show the fluorescence intensity, which was measured in the three clustering examples shown in the top fluorescence image panels, 5 minutes after laminin-1 addition. Laminin-1 induced focal aggregation of BODIPY-GM1 and TrkA in the membrane. The numbered lines in the top image panels correspond to graphs 1-3. Scale bar, 5 μm.

• Supplemental Figure S3 -

  Fig. S3. Laminin-1 directly induced clustering of GM1. Latex beads (5 μm) were coated with 20 μg/ml laminin-1 or 20 μg/ml biotin-transferrin. PC12 cells were labeled with 500 nM BODIPY-GM1, and the coated beads were added to the cells. Images are single confocal sections. BODIPY-GM1 aggregated and colocalized on the cell membrane with laminin-1-coated beads but not with biotin-transferrin-coated beads. Scale bar, 10 μm.

• Supplemental Figure S4 -

  Fig. S4. β1 Integrin relocates to lipid rafts in the presence of NGF and laminin-1. PC12 cells were treated with 100 ng/ml NGF and/or 5 μg/ml laminin-1. Cells were lysed in 1% Triton X-100 and were fractionated on discontinuous sucrose gradients. Raft (R) and non-raft (NR) fractions were collected and subjected to immunoblotting using the indicated antibodies. The bar graph shows the mean + s.d. of three experiments. Relative activity, expressed as the ratio of β1 integrin to flotillin 1, was quantified as the fold-increase relative to NGF (defined as 1) (*P<0.05; **P<0.01; two-sided t-test).

• Supplemental Figure S5 -

  Fig. S5. Effect of laminin-1 and CTxB on NGF signaling. (A,B) PC12 cells were incubated with 100 ng/ml NGF or 5 μg/ml laminin-1 (A) or 2 μg/ml of CTxB (B) or both for indicated times. Phosphorylated TrkA was immunoprecipitated using anti-Trk antibody and analyzed by immunoblotting with anti-phosphorylated-tyrosine antibody. Phosphorylated MAPK and phosphorylated Akt were detected by immunoblotting using specific antibodies. The same blot was stripped and reprobed with anti-TrkA, anti-MAPK and anti-Akt antibodies. The bar graph shows the mean + s.d. of three experiments. Relative activities, expressed as the ratio of phosphorylated TrkA, phosphorylated MAPK or phosphorylated Akt to total TrkA, MAPK or Akt, were quantified as the-fold increase relative to 0 minutes (defined as 1) (*, P<0.05; two-sided t-test).

• Supplemental Figure S6 -

  Fig. S6. Effect of depletion of either β1 integrin or inhibition of GM1 clustering on laminin-1-induced neurite outgrowth. PC12 cells were transfected with vector expressing EGFP and with siRNA targeting β1 integrin (siβ1) or a negative-control siRNA. At 24 hours after transfection with each siRNA, cells transfected with negative-control siRNA were treated with 2.5 μM lyso-GM1 and incubated with 5 μg/ml laminin-1 for 24 hours in the presence of 100 ng/ml of NGF; cells transfected with siRNA targeting β1 integrin were incubated with 5 μg/ml laminin-1 for 24 hours in the presence of 100 ng/ml NGF. Cells were then photographed at the indicated times. Scale bar, 10 μm.