Plexin a1

For Western blot analysis, cells were lysed using lysis buffer containing 10 mM Tris/HCl (PH 7.5), 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 1 mM Na₃CO₄, 1 mM PMSF, and 0.1 mM aprotinin. The cells were scraped from the plates and centrifuged at 16,060 g for 30 min at 4°C. For nuclear protein extraction, the pellet from the 700 g centrifugation was washed by buffer A, resuspended in buffer B containing 20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 2 mM Na₃VO₄, 1 mM pyrophosphoric acid, and Complete TM protease inhibitors (Cell Signaling, USA) and incubated on ice for 5 min. The protein concentration in the cell lysates was determined using the Bradford protein assay. Western blot analysis was performed as previously described using the several antibodies (Bennett et al., 2005 (link)). Sema3A, plexin A1, and NRP1 antibodies were obtained from ABcam, Cambridge, UK. PLCγ2, phospho-PLCγ2, β-cateinin, and DAP12 antibodies were from Cell Signaling, USA.

Cells were seeded on Lab‐Tek Permanox slides overnight, and then treated with appropriate peptide 10⁻⁷ M for 1 h or transfected with the appropriate siRNA for 48 h. After fixation with 1% PFA for 10 min, slices were permeabilized with PBS/0.1% Triton X‐100. Primary antibodies (NRP1 from Evitria, 1:500; and Plexin‐A1 from Abcam, ab23391, 1:200) were incubated overnight at 4°C in PBS. The proximity ligation assay was then performed according to the manufacturer's recommendations with the “detection orange” kit (Sigma). Quantification of the dots was performed using ImageJ software.