Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2010 Apr 14.

Published in final edited form as: Biochemistry. 2009 Apr 14;48(14):3230–3238. doi: 10.1021/bi802163p

Fig. 2 — A. Soluble forms of CIRL GPS mutants. COS cells were transfected with either wild-type CIRL or its GPS mutants with single residue substitution within the GPS domain (C₈₃₄/W, W₈₁₅/S, and T₈₃₈/P), schematically described at the top panel, PM - plasma membrane. The cells were harvested and analyzed by Western blotting with anti-p120 or anti-p85 antibody. The conditioned media were precipitated with α-latrotoxin-agarose followed by Western blotting with anti-p120 antibody. Salmon sperm DNA transfected cells (SS) were used as control. B. Secretion of soluble GPS mutants of the CIRL ectodomain. COS cells were transfected with the plasmids encoding either the wild type CIRL ectodomain or three single-residue mutant constructs (C₈₃₄/W, W₈₁₅/S, and T₈₃₈/P), schematically described at the top panel. The conditioned media were precipitated with α-latrotoxin-agarose followed by Western blotting with either anti-p120 or anti-myc antibody. Note an increase in the apparent size of p120 in the non-cleaved mutants W₈₁₅/S, and T₈₃₈/P due to the 3.8 kDa myc-tag addition. The pictures shown are representative of five independent transfection, precipitation and blotting experiments that produced essentially similar results.

Fig. 2 — A. Soluble forms of CIRL GPS mutants. COS cells were transfected with either wild-type CIRL or its GPS mutants with single residue substitution within the GPS domain (C₈₃₄/W, W₈₁₅/S, and T₈₃₈/P), schematically described at the top panel, PM - plasma membrane. The cells were harvested and analyzed by Western blotting with anti-p120 or anti-p85 antibody. The conditioned media were precipitated with α-latrotoxin-agarose followed by Western blotting with anti-p120 antibody. Salmon sperm DNA transfected cells (SS) were used as control. B. Secretion of soluble GPS mutants of the CIRL ectodomain. COS cells were transfected with the plasmids encoding either the wild type CIRL ectodomain or three single-residue mutant constructs (C₈₃₄/W, W₈₁₅/S, and T₈₃₈/P), schematically described at the top panel. The conditioned media were precipitated with α-latrotoxin-agarose followed by Western blotting with either anti-p120 or anti-myc antibody. Note an increase in the apparent size of p120 in the non-cleaved mutants W₈₁₅/S, and T₈₃₈/P due to the 3.8 kDa myc-tag addition. The pictures shown are representative of five independent transfection, precipitation and blotting experiments that produced essentially similar results.