Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: Pulldown of proCTSD with PGRN expressed in HEK293FT cells. Immunoprecipitation was performed against the FLAG epitope on FLAG-tagged PGRN. Immunoblotting was performed with anti-FLAG and anti-CTSD antibodies. Immunoblots are representative of three independent experiments.

Article Snippet: The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Immunoprecipitation, FLAG-tag, Western Blot

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) Differential scanning fluorimetry (DSF) was used to obtain fluorescent intensity curves versus temperature, and the curve derivatives are plotted for recombinant proteins: 1.5μM HIS-tagged proCTSD alone (blue), 4.5μM HIS-tagged PGRN alone (black), and 1.5μM proCTSD with 4.5μM PGRN (red). DSF was performed at neutral pH 7.4. Assays were run in triplicate and values plotted are mean ± SEM. (B) Melting temperature, Tm, for PGRN:proCTSD complex at increasing molar ratios of PGRN.

Article Snippet: The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Recombinant

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A-D) In vitro maturation time-course assays for recombinant HIS-tagged proCTSD at pH 3.4 in the (A) absence (n = 4) and presence of (B) 75nM (n = 3), (C) 100nM (n = 3) and (D) 150nM (n = 3) HIS-tagged PGRN. Membranes were immunoblotted with anti-CTSD antibody. Boxed regions in panel B indicate the signal measured for quantification of % 30kDa CTSD / total. (E) Michaelis-Menten kinetic curves for the conversion of proCTSD to matCTSD. (F) Correlation between initial velocity of reaction (V0) and PGRN concentration.

Article Snippet: The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: In Vitro, Recombinant, Concentration Assay

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) ProCTSD undergoes an autocatalytic activation mechanism for the formation of initial matCTSD molecules at a low rate. (B) Once formed, matCTSD can convert proCTSD to matCTSD through an intermolecular cleavage of the propeptide. (C) PGRN binds around the propeptide region of proCTSD to destabilize its interaction with the enzyme catalytic core, (D) facilitating propeptide cleavage by matCTSD. Catalytic aspartyl residues are represented as orange dots, the propeptide in blue and the propeptide cleavage site in red.

Article Snippet: The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Activation Assay

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: Pulldown of proCTSD with PGRN expressed in HEK293FT cells. Immunoprecipitation was performed against the FLAG epitope on FLAG-tagged PGRN. Immunoblotting was performed with anti-FLAG and anti-CTSD antibodies. Immunoblots are representative of three independent experiments.

Article Snippet: HEK293FT transfections and co-immunoprecipitation assays The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Immunoprecipitation, FLAG-tag, Western Blot

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) Differential scanning fluorimetry (DSF) was used to obtain fluorescent intensity curves versus temperature, and the curve derivatives are plotted for recombinant proteins: 1.5μM HIS-tagged proCTSD alone (blue), 4.5μM HIS-tagged PGRN alone (black), and 1.5μM proCTSD with 4.5μM PGRN (red). DSF was performed at neutral pH 7.4. Assays were run in triplicate and values plotted are mean ± SEM. (B) Melting temperature, Tm, for PGRN:proCTSD complex at increasing molar ratios of PGRN.

Article Snippet: HEK293FT transfections and co-immunoprecipitation assays The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Recombinant

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A-D) In vitro maturation time-course assays for recombinant HIS-tagged proCTSD at pH 3.4 in the (A) absence (n = 4) and presence of (B) 75nM (n = 3), (C) 100nM (n = 3) and (D) 150nM (n = 3) HIS-tagged PGRN. Membranes were immunoblotted with anti-CTSD antibody. Boxed regions in panel B indicate the signal measured for quantification of % 30kDa CTSD / total. (E) Michaelis-Menten kinetic curves for the conversion of proCTSD to matCTSD. (F) Correlation between initial velocity of reaction (V0) and PGRN concentration.

Article Snippet: HEK293FT transfections and co-immunoprecipitation assays The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: In Vitro, Recombinant, Concentration Assay

Journal: Journal of molecular biology

Article Title: Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH

doi: 10.1016/j.jmb.2019.01.027

Figure Lengend Snippet: (A) ProCTSD undergoes an autocatalytic activation mechanism for the formation of initial matCTSD molecules at a low rate. (B) Once formed, matCTSD can convert proCTSD to matCTSD through an intermolecular cleavage of the propeptide. (C) PGRN binds around the propeptide region of proCTSD to destabilize its interaction with the enzyme catalytic core, (D) facilitating propeptide cleavage by matCTSD. Catalytic aspartyl residues are represented as orange dots, the propeptide in blue and the propeptide cleavage site in red.

Article Snippet: HEK293FT transfections and co-immunoprecipitation assays The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020).

Techniques: Activation Assay