Journal: International Journal of Molecular Sciences
Article Title: Calsenilin, a Presenilin Interactor, Regulates RhoA Signaling and Neurite Outgrowth
Figure Lengend Snippet: Calsenilin-CTF is responsible for the interaction with RhoA: ( A ) schematic diagram depicting the construction of the pEGFP-N1 vector, calsenilin-FL (aa 1–256), -NTF (aa 1–64), and -CTF (aa 65–256); ( B , C ) total lysates from HEK293 and PC12 cells expressing pEGFP-N1 vector alone (VEC), full-length calsenilin (FL), or truncated fragments (NTF or CTF) were immunoprecipitated with an anti-RhoA antibody and then analyzed by Western blotting with anti-calsenilin (1F11, amino acid 25-33; 4E4), anti-p190RhoGAP and anti-RhoA antibodies; and ( D ) the co-localization of calsenilin with RhoA in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF was determined by double immunofluorescence staining using confocal microscopy. Green, calsenilin; Red, RhoA; Blue, DAPI. Scale bars, 20 μm.
Article Snippet: The calsenilin–EGFP constructs were made by inserting the FL, NTF, or CTF of calsenilin into the pEGFP-N1 vector (BD Biosciences, Mississauga, ON, Canada) after amplifying the sequences from an existing pcDNA3.1/Zeo(+) calsenilin with the following primers (the endonuclease sites are underlined): FL-EGFP, forward 5′-CACTCGAGGCCACCATGCAGCCGGCTAAGGAA-3′ (XhoI) and reverse 5′-GAGAAGCTTGATGACATTCTCAAACTG-3′ (HindIII); NTF-EGFP, forward 5′-CACTCGAGGCCACCATGCAGCCGGCTAAGGAA-3′ (XhoI) and reverse 5′-GAGAAGCTTGTCGCTGCTATCTGAGCC-3′ (HindIII); and CTF-EGFP, forward 5′-CACTCGAGGCCACCATGAGTGAGCTGGAGCTG-3′ (XhoI) and reverse 5′-GAGAAGCTTGATGACATTCTCAAACTG-3′ (HindIII).
Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Double Immunofluorescence Staining, Confocal Microscopy