Journal: Atherosclerosis Plus

Article Title: Characterization of selected LDLR substitutions in patients with familial hypercholesterolemia

doi: 10.1016/j.athplu.2025.11.001

Figure Lengend Snippet: Cellular expression of the LDLR variants. HEK293T- ldlr G1 cells transfected with pTetRedLDLR expression vectors were cultured in the medium supplemented with 100 ng/mL doxycycline for 48 h. The cell lysates were loaded onto a gel at equal total protein concentrations and analyzed by Western blot. A: The 150 kDa bands represent mature form of LDLR wild-type (WT) (line 1), c.91G > A p. (Glu31Lys) (line 2), c.662 A > G p. (Asp221Gly) (line 3), c.1775G > A p. (Gly592Glu) (line 4), c.2483delA p. (Tyr828Phefs∗101) (line 5), c.661G > T p. (Asp221Tyr) (line 6), c.1216C > T p. (Arg406Trp) (line 7), c.2177C > T p. (Thr726Ile) (line 8), Hek293T- ldlr G1 non-transfected (line 9), respectively. The 25 kDa signals represent the DsRed2 protein, indicating the correct transfection process. The 50 kDa bands demonstrate β-actin. B: LDLR signals were normalized to the intensity of DsRed2. The relative level of LDLR variants was assessed using the ratio of the variants compared to wild-type band intensity. The mean of four independent Western blots (n = 4) is shown in the graph. The green line is the 90 % threshold, and the red line is the 70 % threshold. Asterisks indicate statistically significant results in the T-test.

Article Snippet: The surface expression of LDLR was detected using a mouse monoclonal anti-LDLR primary antibody (1:100) (Cat. No. 61087, Progen Biotechnik GmbH, Heidelberg; Germany) followed by an Alexa FluorTM 488- conjugated goat anti-mouse secondary antibody (1:2000) (Cat. No. A11001, Thermo Fisher Scientific; Waltham; MA; USA).

Techniques: Expressing, Transfection, Cell Culture, Western Blot

Journal: Atherosclerosis Plus

Article Title: Characterization of selected LDLR substitutions in patients with familial hypercholesterolemia

doi: 10.1016/j.athplu.2025.11.001

Figure Lengend Snippet: Analysis of LDLR variant expression on the cell membrane by confocal microscope. A-C: Transfected cells immunostained with anti-LDLR and Alexa Fluor™ 488 goat anti-mouse IgG conjugated antibodies. The green fluorescence intensity indicated the receptor expression level on a cellular surface. The yellow fluorescence of DsRed2 monitored transfected cells. The nucleus stained with PureBlu™ Hoechst 33342 was visible as blue fluorescence. D: The percentage of LDLR was represented on the graph as the mean ± SD (n = 30 cells). The green line is the 90 % threshold, and the red line is the 70 % threshold. Asterisks indicate statistically significant results in the T-test.

Article Snippet: The surface expression of LDLR was detected using a mouse monoclonal anti-LDLR primary antibody (1:100) (Cat. No. 61087, Progen Biotechnik GmbH, Heidelberg; Germany) followed by an Alexa FluorTM 488- conjugated goat anti-mouse secondary antibody (1:2000) (Cat. No. A11001, Thermo Fisher Scientific; Waltham; MA; USA).

Techniques: Variant Assay, Expressing, Membrane, Microscopy, Transfection, Fluorescence, Staining

Journal: Atherosclerosis Plus

Article Title: Characterization of selected LDLR substitutions in patients with familial hypercholesterolemia

doi: 10.1016/j.athplu.2025.11.001

Figure Lengend Snippet: Internalization capacity of LDLR variants. A: HEK293T- ldlr G1 cells transfected with pTetRedLDLR expression vectors were incubated with the pHrodo™ Green-LDL. LDL uptake was observed in a confocal microscope as increasing green fluorescence over time. Yellow fluorescence of DsRed2 indicated transfected cells. B: The increment of internalization capacity of LDLR variants over time is presented as the percentage mean ± SD (n = 50 cells) compared to WT. Asterisks indicate statistically significant results in the T-test. C: LDL uptake rate constant. The green line is the 90 % threshold, and the red line is the 70 % threshold.

Article Snippet: The surface expression of LDLR was detected using a mouse monoclonal anti-LDLR primary antibody (1:100) (Cat. No. 61087, Progen Biotechnik GmbH, Heidelberg; Germany) followed by an Alexa FluorTM 488- conjugated goat anti-mouse secondary antibody (1:2000) (Cat. No. A11001, Thermo Fisher Scientific; Waltham; MA; USA).

Techniques: Transfection, Expressing, Incubation, Microscopy, Fluorescence

Journal: Atherosclerosis Plus

Article Title: Characterization of selected LDLR substitutions in patients with familial hypercholesterolemia

doi: 10.1016/j.athplu.2025.11.001

Figure Lengend Snippet: Measurement of ligand binding capacity by LDLR variants. A: HEK293T- ldlr G1 cells transfected with pTetRedLDLR expression vector were fixed and incubated with BODIPY™ FL-LDL. The LDL binding to receptors was observed in a confocal microscope as the intensity of green fluorescence. The yellow fluorescence of DsRed2 monitored transfected cells. The nucleus stained with PureBlu™ Hoechst 33342 was visible as blue fluorescence. B: Ligand binding capacity represents the percentage mean ± SD (n = 65 cells). Asterisks indicate statistically significant results (p < 0.05) in the T-test. The green line is the 90 % threshold, and the red line is the 70 % threshold.

Article Snippet: The surface expression of LDLR was detected using a mouse monoclonal anti-LDLR primary antibody (1:100) (Cat. No. 61087, Progen Biotechnik GmbH, Heidelberg; Germany) followed by an Alexa FluorTM 488- conjugated goat anti-mouse secondary antibody (1:2000) (Cat. No. A11001, Thermo Fisher Scientific; Waltham; MA; USA).

Techniques: Ligand Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Binding Assay, Microscopy, Fluorescence, Staining