rabbit polyclonal antibodies against pcsk9  (Bioss)

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Generation of PCSK9_55 in cells culture. (A) Extracts and conditioned media of HEK293T cells transfected with empty vector (lanes 2 and 5) or with pPCSK9 62 (lanes 3 and 6) were separated via SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lanes 1 and 4). Image is representative of 3 independent experiments. (B) Total PCSK9 quantified in the intracellular and extracellular compartments of our in vitro conditions in HEK293T cells transfected with pPCSK9 62. (C) Experimental strategy followed in cell culture to generate PCSK9_55 under semiphysiologic conditions cotransfecting pPCSK9 62 and pFURIN. (D) Extracts and conditioned media from HEK293T cells transfected with pPCSK9 62 and empty vector (lanes 2 and 4) or pPCSK9 62 and pFURIN (lanes 3 and 5) were separated on an SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lane 1). (E) Quantification of PCSK9 bands from “D” are plotted relative to pPCSK9 62 in the cell extracts and cell media. Image is representative of ≥13 independent experiments. Values are mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (∗) Cell media is loaded with unequal protein load as in cell extracts. Dotted lines indicate splicing of original image.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: Transfection, Plasmid Preparation, SDS Page, In Vitro, Cell Culture

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Furin inhibition selectively limits PCSK9_62 conversion to PCSK9_55 in the extracellular space. HEK293T cells cotransfected with pPCSK9 62 and pFURIN were treated with the cell-permeable furin inhibitor dec-RVKR-cmk (25 μM) to repress PCSK9 cleavage. (A) Extracts and conditioned media of HEK293T cells transfected with pPCSK9 62 and empty vector (lanes 2 and 6, and treated with furin inhibitor lanes 4 and 8) or pPCSK9 62 and pFURIN (lanes 3 and 7, and treated with furin inhibitor lanes 5 and 9) were separated via SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lane 1). (B) Quantification of PCSK9 bands from “A” are plotted relative to pPCSK9 62 in the cell extracts and cell media. Image is a representative of 4 independent experiments. Values are mean ± SEM. ∗ P < 0.05. (∗) Cell media is loaded with unequal protein load as in cell extracts. Dotted lines indicate splicing of the original image.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: Inhibition, Transfection, Plasmid Preparation, SDS Page

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Kinetics of PCSK9_62 conversion to PCSK9_55 in vitro. Pulse-chase experiments traced the rate of PCSK9 production intracellularly and the rate of secretion to the extracellular space. Storage phosphor screen scans show PCSK9 presence over time (0–22 h) in cell extracts (A) and media (B) of HEK293T cells cotransfected with pPCSK9 62 and empty vector (empty circles) or pPCSK9 62 and pFURIN (full circles). Quantification of PCSK9_62 bands from “A” are plotted relative to pPCSK9 62 and empty vector condition at time 0 h, whereas for “B”, the 7 kDa peptide bands are plotted relative to pPCSK9 62 condition and empty vector at time 1 h. Image is a representative of 3 independent experiments. Values are mean ± SEM. Cells transfected with pcDNA3 were used as a negative control, and pPCSK9 55 was used as a reference to identify intracellular PCSK9_55.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: In Vitro, Pulse Chase, Plasmid Preparation, Transfection, Negative Control

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Intracellular PCSK9_55 fails to be secreted to the extracellular space. (A) Experimental strategy followed to characterize the engineered PCSK9_55 in cell culture. (B) Extracts and conditioned media of HEK293T cells transfected with pPCSK9 62 (lanes 2 and 4) or pPCSK9 55 (lanes 3 and 5) were separated via SDS-PAGE and immunoblotted for PCSK9 and actin (MW ladder; lane 1). Image is representative of ≥11 independent experiments. (C) Quantification of PCSK9 bands from “B” are plotted relative to pPCSK9 62 in the cell extracts and cell media. (D) Extracts and conditioned media of HEK293T cells transfected with pPCSK9 62 and empty vector (lanes 2 and 9), pPCSK9 PD and empty vector (lanes 3 and 10), pPCSK9 ΔPD and empty vector (lanes 4 and 11), pPCSK9 55 and empty vector (lanes 5 and 12), pPCSK9 55 and pPCSK9 PD (lanes 6 and 13), and pPCSK9 ΔPD and pPCSK9 PD (lanes 7 and 14) were separated via SDS-PAGE and immunoblotted for PCSK9 (MW ladder; lanes 1 and 8). Image is representative of 2 independent experiments. (E) Extracts and conditioned media of HEK293T cells transfected with pPCSK9 62 and empty vector (lanes 2 and 6), pPCSK9 55 and empty vector (lanes 3 and 7), and pPCSK9 62 and pPCSK9 55 (lanes 4 and 8) were separated via SDS-PAGE and immunoblotted for PCSK9 (MW ladder; lanes 1 and 5). Image is representative of 1 independent experiment. Values are mean ± SEM. ∗∗∗ P < 0.001. (∗) Cell media is loaded with unequal protein load as in cell extracts. Dotted lines indicate splicing of the original image.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: Cell Culture, Transfection, SDS Page, Plasmid Preparation

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Development and validation of a functional assay to quantify PCSK9 activity on cellular LDLRs. LDLR levels in HEK293T cells are monitored by flow cytometry. (A) Representative plots that capture LDLR content as the percentage of GFP-positive cells (forward scatter height against GFP signal) in cells transfected with an empty vector (left plot), transfected with an LDLR-GFP-tagged expression vector and incubated with a vehicle (center) and transfected with an LDLR-GFP-tagged expression vector and incubated with a recombinant PCSK9_62 (3 μg/mL) for 24 h (right). (B) Quantification of LDLR levels upon incubation with recombinant PCSK9_62, as in “A.” (C) Quantification of LDLR levels upon transfection with pPCSK9 62 or pPCSK9 55. (D) Cell extracts (same conditions as in “C”) after separation by SDS-PAGE and immunoblotting for LDLR, PCSK9, and actin. Images are representative of 2 independent experiments. Data are representative of > 4 independent experiments that were run in duplicate or triplicate. Values are mean ± SEM. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. Dotted lines indicate splicing of the original image.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: Biomarker Discovery, Functional Assay, Activity Assay, Flow Cytometry, Transfection, Plasmid Preparation, Expressing, Incubation, Recombinant, SDS Page, Western Blot

Journal: Journal of Lipid Research

Article Title: Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9

doi: 10.1194/jlr.RA120000964

Figure Lengend Snippet: Working model. Newly synthesized PCSK9 undergoes an autocatalytic cleavage inside the cell that releases the prodomain (13 kDa) from the peptide chain. (A) The cleaved prodomain binds back to the main protein through noncovalent forces generating a heterodimer of 62 + 13 kDa, representing the mature form of PCSK9 (PCSK9_62). (B) This is an essential step for the proper secretion of PCSK9_62 into circulation. PCSK9_62 can also undergo a second cleavage, in the extracellular space, mediated by the protease furin. (C) Furin cleaves PCSK9 at the N-terminal region releasing an ∼7 kDa peptide and potentially the prodomain, generating the second most common form of plasma PCSK9 with a size of 55 kDa (PCSK9_55). (D) Once in circulation, both forms of PCSK9 induce hepatic LDLR degradation, though PCSK9_62 is more active than PCSK9_55. (E) PCSK9 elimination from circulation is mediated by LDLR-dependent and LDLR-independent pathways. Potentially, the furin-cleaved form of PCSK9, PCSK9_55, has a shorter half-life owing to a faster clearance rate than PCSK9_62. (F) Once inside the cell, PCSK9_55 cannot get secreted back to circulation and (G) before going through catabolism, (H) the intracellular pool of PCSK9_55 is capable of inducing LDLR degradation though less so relative to PCSK9_62.

Article Snippet: In brief, 5 μg of rabbit polyclonal antibody against human PCSK9 (Cat# CY-1307, MBL international) was coupled to protein G-coated magnetic beads and cross-linked with 5 mM BS3 (Cat # 21580, Thermo Fisher Scientific).

Techniques: Synthesized, Clinical Proteomics