Fig. 1

PC12 stable transfectants expressing RFP or RFP-zRICH(H334A) fusion proteins. A: Structure of the proteins expressed in PC12 cells. The protein designated as RFP is 251 amino acids long and it is composed of the DsRed-monomer RFP fused to a peptide derived from the polylinker region of the plasmid. The 672 amino acid long fusion protein RFP-zRICH(H334A) consists of the full size 424 amino acid long zRICH(H334A) fused to the amino-terminal 248 amino acid long fragment derived from RFP. The H to A amino acid substitution at position 334 of the zRICH protein portion, affecting a key catalytic site residue within the CNPase homology domain (light green shade) is indicated in the diagram. B: Western blot analysis of PC12 stable transfectants. Immunodetection was performed with anti-RICH antibody (top-left membrane) or anti-RFP antibody (top-right membrane). Both anti-RICH and anti-RFP detected the expressed RFP-zRICH(H334A) fusion protein (apparent molecular weight of approximately 100 kDa). The much smaller RFP protein was detected in the lysate from PC12-RFP cells above the 30 kDa size marker. A degradation product detected by anti-RFP just above 30 kDa is present in both of the stable transfectants. Untransfected PC12 cells were used as negative control and 100 ng of recombinant zRICH(WT) was used to confirm antibody specificity and estimate levels of expression of the fusion protein by densitometric analysis (35 ng on the membrane, 0.7 ng/µg of total cell protein). Immunoblots with anti-Tubulin antibody (bottom panels) confirmed that similar amounts of protein lysates from the cells were utilized. Images were cropped to facilitate visualization (using GIMP software). Original unprocessed images of full-length blots are presented in Supplementary Material 7