Figure 5: KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation.

(a) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml⁻¹) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). (b) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. (c) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. (d) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. (e) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).