anti pcsk9 d7u6l rabbit monoclonal primary antibody  (Cell Signaling Technology Inc)

Journal: Molecular Biology Reports

Article Title: HepG2 cells stimulated by THP-1-conditioned medium: a potential in vitro model of systemic inflammation–induced hepatic alterations

doi: 10.1007/s11033-026-11641-0

Figure Lengend Snippet: Time-dependent changes in the relative mRNA expression of the inflammatory markers NF-κB p50/p105, TNF-α, A20, and SAA ( a ), transcription factors PPARα and LRH-1 ( b ), and lipid metabolism–associated genes LDLr, PON1, PCSK9, ApoA1, ABCA1, and ApoC3 ( c ) in HepG2 cells stimulated with CM (diluted 1:3) derived from THP-1 cells activated with PMA + LPS. Time-course experiments were performed in three independent biological experiments with duplicates for most time points. For selected key time points (4 h and 24 h), which were identified as the most relevant based on previous experiments, an additional experiment with biological quadruplets was conducted to increase precision. Protein expression of ApoA1 and PCSK9 ( d ) in HepG2 cells 24 h after stimulation with CM. Data represent nine biological replicates from three independent experiments. * p < 0,05 ** p < 0,01 *** p < 0,001 **** p < 0,0001

Article Snippet: Western blot was performed using standard protocol with primary antibodies against β-actin (Abcam, Cambridge, UK, EPR21241 ), ApoA1 (Abcam, Cambridge, UK, EP1368Y) and PCSK9 (Cell Signalling Technology, Netherlands, 85813T), and secondary antibodies conjugated to horseradish peroxidase.

Techniques: Expressing, Derivative Assay

Journal: Molecular Biology Reports

Article Title: HepG2 cells stimulated by THP-1-conditioned medium: a potential in vitro model of systemic inflammation–induced hepatic alterations

doi: 10.1007/s11033-026-11641-0

Figure Lengend Snippet: Relative mRNA expression of inflammatory markers, transcription factors and lipid metabolism–associated genes in HepG2 cells after stimulation with CM derived from THP-1 cells treated with PMA + LPS (CM) or from THP-1 cells that were left non-stimulated (NS). mRNA was isolated at suitable time points based on previous experiments: 4 h for NF-κB p50/p105, TNF-α, A20, SAA, PPARα, LRH-1, and LDLr ( a ) and 24 h for PON1, PCSK9, ApoA1, ABCA1, and ApoC3 ( b ). qPCR analyses represent the average of three independent biological experiments. In the first experiment, three biological replicates were analysed. The second and third experiments each involved four biological replicates.* p < 0,05 ** p < 0,01 *** p < 0,001 **** p < 0,0001

Article Snippet: Western blot was performed using standard protocol with primary antibodies against β-actin (Abcam, Cambridge, UK, EPR21241 ), ApoA1 (Abcam, Cambridge, UK, EP1368Y) and PCSK9 (Cell Signalling Technology, Netherlands, 85813T), and secondary antibodies conjugated to horseradish peroxidase.

Techniques: Expressing, Derivative Assay, Isolation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Engineering chimeric PCSK9 for a vaccine against atherosclerosis

doi: 10.1016/j.omtm.2025.101535

Figure Lengend Snippet: PCSK9 chimera design process with protein characterization and proof-of-concept BALB/c immunization, with serum endpoint titers and splenocyte (T cell) reaction assay (A) The algorithm, showing the design process to determine chimera sequence, the chimera model (chPCSK9), and the model of the mature hPCSK9 chain (PDB: 6U26 ). (B) SEC-MALS of purified recombinant human (hPCSK9, gray) and chimeric human (chPCSK9, blue) proteins, exhibiting the molar mass (74.81 kDa for hPCSK9 and 52.95 kDa for chPCSK9) and protein integrity (single peak). hPCSK9 is present with the bound prodomain, whereas chPCSK9 consists only of the mature chain. (C) The diagram of the vaccination regime shows three consecutive injections: priming, followed by two boosters 2 weeks apart. The relevant organs (spleen, liver, muscles) were harvested with the final blood. (D) Endpoint titers of total IgG antibodies against hPCSK9, determined by ELISA. The overall statistical significance for each time point was calculated using a two-way ordinary ANOVA, comparing all immunization groups to hPCSK9 group. Each immunization group is shown separately (hPCSK9: blue, chPCSK9: red, chPCSK9-tox: orange, chmPCSK9 (chimeric mouse): green, mPCSK9 (mouse): beige); individual dots represent each animal within the immunization group. n = 6 for all immunization groups except for chPCSK9-tox, where n = 4. (E) Endpoint titers of total IgG antibodies against mPCSK9, determined by ELISA. The overall statistical significance for each time point was calculated using a two-way ordinary ANOVA, comparing all immunization groups to hPCSK9 group. Each immunization group is shown separately (hPCSK9: blue, chPCSK9: red, chPCSK9-tox: orange, chmPCSK9: green, mPCSK9: beige); individual dots represent each animal within the immunization group. n = 6 for all immunization groups except for chPCSK9-tox, where n = 4. (F and G) ELISA showing quantification of mIFNγ (E) and mIL-2 (F) released from isolated splenocytes upon stimulation with 50 μg/mL recombinant hPCSK9 or mPCSK9, indicated on the x axis. Significance was calculated using an ordinary two-way ANOVA; results are shown as mean ± SEM of each immunization group, colored same as in D. (H) Unstimulated (fresh) splenocyte flow cytometry analysis, showing the presence of CD3 + T cells in each immunization group (colored as in D and 1E). Significance was calculated using ordinary one-way ANOVA. Each dot represents each animal within the immunization group. (I) Unstimulated splenocyte flow cytometry analysis of T cell subsets on the x axis (naive CD4 − CD8 − , T helper cells CD4 + CD8 − , cytotoxic T cells CD4 − CD8 + ) in each immunization group (colored as in D and 1E). The statistical significance was calculated using ordinary one-way ANOVA. Each dot represents each animal within the immunization group. (J) Unstimulated (fresh) splenocyte flow cytometry analysis of IFNγ + CD8 + T cells in each immunization group (colored as in D and 1E). Significance was calculated using an ordinary two-way ANOVA. Each dot represents each animal within the immunization group.

Article Snippet: Anti-LDLR (Sino Biological, cat. no. 50305-R032, clone 032) and anti-PCSK9 antibodies (Cell Signaling, cat. no. 85813S, clone D7U6L) were used for the quantification of the respective proteins; anti αβ-tubulin antibodies (Cell Signaling, Cat. No. 2148, polyclonal) were used as loading control.

Techniques: Sequencing, Purification, Recombinant, Muscles, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Engineering chimeric PCSK9 for a vaccine against atherosclerosis

doi: 10.1016/j.omtm.2025.101535

Figure Lengend Snippet: Atherosclerosis development in aortic arches and adjacent tissues of ApoE−/− mice, vaccinated with human chimeric PCSK9 (chPCSK9-tox) (A) Violin plot of ORO staining area results of aortic arch histology slides, obtained using the deconvolution approach in ImageJ. The red data represent control group; the yellow data represent chPCK9-tox. Significance was calculated using the unpaired Welch’s t test. (B) Aortic widths of the aortic arch histology slides, measured in ImageJ with the measuring tool. The dark blue data represents the control group; the light blue data represent the chPCK9-tox group. Significance was calculated using the unpaired t test. (C) Representative micrographs of the aortic arch of control (pCG1) and chPCSK9-tox groups. Red arrows show ORO-stained areas, and blue arrows show representative width measurements. The scale used for all images is 250 μm. (D) Representative confocal microscopy images of aortic walls, showing presence of Mac-3 (CD107b; magenta) with Hoechst 33342 (cyan) as counterstain for the nuclei. The scale used is 50 μm for the first two rows and 25 μm for the last row. Pink arrows indicate developing atherosclerotic plaques on the aortic walls.

Article Snippet: Anti-LDLR (Sino Biological, cat. no. 50305-R032, clone 032) and anti-PCSK9 antibodies (Cell Signaling, cat. no. 85813S, clone D7U6L) were used for the quantification of the respective proteins; anti αβ-tubulin antibodies (Cell Signaling, Cat. No. 2148, polyclonal) were used as loading control.

Techniques: Staining, Control, Confocal Microscopy