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. 2015 Sep 15;4:e07068. doi: 10.7554/eLife.07068

Figure 2. Nrp1 interacts with PDE4D, and Sema3 promotes this interaction.

(A) Immunoblot showing that the endogenous PDE4D isoform 2/6 from NIH3T3 cells was immunoprecipated by Nrp1 antibody, but not by an unrelated GFP antibody. (B, C) Immunoblots showing the amount of PDE4D2/6 immunoprecipitated by Nrp1 antibody from cells treated with Sema3 (a combination of Sema3A+3F) for different periods of time. (B). Band intensity was normalized to the input at the corresponding time point, and then normalized to time 0 (C). (D, E) Immunoblots showing the amount of Nrp1 and PDE4D2/6 in the membrane and cytosolic fractions from cells treated with Sema3A+3F for different periods of time. (D). Band intensity was normalized to time 0 (E). Quantification in (C) and (E) were from three independent experiments. Error bars represent SEM. Statistics: Student's t-Test. *p < 0.05, **p < 0.01.

DOI: http://dx.doi.org/10.7554/eLife.07068.005

Figure 2.

Figure 2—figure supplement 1. The cytoplasmic domain of Nrps mediates their interactions with PDE4D2.

Figure 2—figure supplement 1.

(A) Major PDE4D isoforms expressed in NIH3T3 cells recognized by a PDE4D antibody against the consensus C-terminus of all PDE4D isoforms. (B, C) Flag- tagged PDE4D2 co-immunoprecipitates with full length EGFP-tagged Nrp1/2 after they are overexpressed in NIH3T3 cells, but not with cytoplasmic domain-truncated Nrps (Nrp1/2-Δcyto). (D) Immunoblots showing the amount of Nrp1/2 and PDE4D2/6 in the membrane fractions from cells treated with Shh, Sema3A+3F, or the combination of Shh and Sema3s for 30 min. (E) Band intensity was normalized to vehicle. (F) After Nrp1+2 were silenced by RNAi, membrane fractions were isolated from the cell. Immunoblots showing the amount of Nrp1/2 and PDE4D2/6 in the membrane fractions from cells treated with Sema3 (3A+3F) for 30 min. (E) Band intensity was normalized to vehicle. Quantification in (E) and (G) were from three independent experiments. Error bars represent SEM. Statistics: Student's t-Test. *p < 0.05, **p < 0.01.
DOI: http://dx.doi.org/10.7554/eLife.07068.006
Figure 2—figure supplement 2. Sema3-Nrp signaling reduces intracellular cAMP levels.

Figure 2—figure supplement 2.

(A) cAMP standard curve generated by cAMP-Glo assay showing the linear relationship between cAMP concentration and the luminescence intensity change (ΔLuminescence). (B) ΔLuminescence was read out after cells were treated with control (BSA), forskolin (1 μM) or Sema3F (3ug/ml) for 30 min ΔLuminescence was calculated as Luminescencetreated—Luminescenceuntreated. (C) Cells were infected with lentivirus expressing shRNA against Nrp1&2 for 72 hr ΔLuminescence was read out after cells were treated with control (BSA), or Sema3F (3ug/ml) for 30 min. Statistics: Non-parametric Mann–Whitney test. All error bars represent SEM. *p < 0.05, **p < 0.01.
DOI: http://dx.doi.org/10.7554/eLife.07068.007