JCS024620 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression level of afadin, nectin-1 and nectin-3 in afadin- or nectin-3-knockdown NIH3T3 cells. Lysates from wild-type, control, afadin-knockdown and nectin-3-knockdown NIH3T3 cells were used for Western blotting with the indicated Abs.

• Supplemental Figure S2 -

  Fig. S2. Colocalization of the nectin-afadin complex with PDGF receptor at cell-cell adhesion sites. Confluent NIH3T3 cells were immunostained with the rabbit anti-PDGF receptor mAb (Abcam) and the rat anti-nectin-3 or mouse anti-afadin mAb. Note that essentially the same results were observed in this figure and in Fig. 6Aa. Scale bars, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. No effect of nectin-1 on apoptosis in NIH3T3 cells. (A) Knockdown of nectin-1. Lysates from wild-type and nectin-1-knockdown NIH3T3 cells were used for western blotting to examine the efficiency of siRNA against nectin-1 for knockdown of nectin-1. (B) No increase in the number of apoptotic nectin-1-knockdown NIH3T3 cells. Confluent control and nectin-1-knockdown NIH3T3 cells were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours in the presence of 3 ng/ml PDGF. Apoptotic cells were detected using the TUNEL method. Nuclei were counterstained with DAPI and are seen in blue. The graph represents the mean percentage of TUNEL-positive cells in three independent experiments. In an experiment, the number of TUNEL-positive cells among a total 100 of cells observed in four different fields of view was counted. Error bars indicate s.e.m. No significant difference was observed between control and nectin-1-knockdown NIH3T3 cells.