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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Mol Cell Neurosci. 2013 Sep 10;57:10.1016/j.mcn.2013.08.004. doi: 10.1016/j.mcn.2013.08.004

Figure 3. Sema4D is cleaved in hippocampal neurons.

Figure 3

(a) Western Blot analysis of endogenous Sema4D protein expression in hippocampal lysates isolated from either wildtype (+/+) or Sema4D knockout (−/−) mice at postnatal day 8 using both polyclonal (pAb) and monoclonal (mAb) antibodies raised against the C-terminus of Sema4D. Full length Sema4D protein runs at approximately 150kD.

(b) Western blot analysis of lysates from rat hippocampal neurons infected with either empty lentiviral vector or lentivirus containing the Sema4D-FLAG construct, probed with monoclonal and polyclonal antibodies which recognize Sema4D, as well as a monoclonal antibody which recognizes the FLAG epitope. The presence of the Sema4D C-terminal cleavage product was detected at approximately 25kD.

(c) Western blot analysis of lysates from rat hippocampal neurons infected with either empty lentiviral vector or lentivirus containing the myc-Sema4D construct probed with monoclonal and polyclonal antibodies which recognize Sema4D, as well as a monoclonal antibody which recognizes the myc epitope. As expected, the presence of the 25kD Sema4D C-terminal cleavage product was not detected with the monoclonal antibody, which recognizes the myc epitope.

(d) Western blot analysis of hippocampal tissue lysates isolated from postnatal day 1 rat pups and subjected to immunoprecipitation with either a polyclonal antibody which recognizes Sema4D, or the corresponding pre-immune sera. Blots are probed with a monoclonal antibody, which recognizes Sema4D.