To determine cell surface expression of LDLR by FACS, transfected CHO-ldlA7 cells grown during 48 h were incubated with a mouse primary antibody anti-LDLR (1∶100; 5 mg/L; Progen Biotechnik GmbH, Cat. No. 61087) for 1 h, at room temperature, then, washed twice with PBS-1%BSA and incubated with a secondary antibody Alexa Fluor 488-conjugated goat anti-mouse IgG (1∶100; Molecular Probes; Cat. No. A-11001). As negative controls, non transfected CHO-ldlA7 cells or transfected with the wt LDLR were stained with the same primary anti-LDLR antibody (Progen Biotechnik GmbH, Cat. No. 61087), which is negative for CHO-ldlA7 cells and, with mouse IgG2b, kappa monoclonal [MPC-11]-isotype control (abcam; Cat. No. ab18457) in identical conditions. As shown in Figure S1 , isotype control antibody has no specificity for CHO-ldlA7 cells transfected or not with LDLR. For each sample, fluorescence of 10,000 events was acquired for data analysis. All measurements have been performed at least in triplicate. LDLR expression efficiency was corrected using the data of mature protein expression quantified by Western blot and data is shown as the percentage of maximum obtained for wt LDLR.
Anti ldlr
Manufactured by Progen Biotechnik
The Anti-LDLR is a laboratory equipment designed to measure the levels of low-density lipoprotein receptor (LDLR) in biological samples. It utilizes advanced detection methods to quantify the presence and concentration of LDLR within the sample.
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2 protocols using anti ldlr
1
Quantifying LDLR Cell Surface Expression by FACS
2
Quantifying LDLR Expression and Internalization
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For detection of the amount of cell-surface LDLR expression, HepG2 cells were harvested using Non-enzymatic Cell Dissociation Solution (Sigma-Aldrich), washed twice with Staining Buffer (PBS þ 1% BSA) and incubated with anti-LDLR (1:20 dilution in Staining Buffer; Progen) at room temperature for 40 min. Cells were then washed three times and incubated with Alexa Fluor 647conjugated anti-mouse (1:600 dilution in Staining Buffer; Abcam) at room temperature for 30 min in the dark. After antibody incubation, cells were washed twice with Staining Buffer, resuspended in PBS and analyzed on a FACS Canto flow cytometer (BD Biosciences) for quantification of Alexa Fluor 647 fluorescence. To measure LDLR internalization activity, human LDL was isolated and labelled with 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine perchlorate (DiD; Invitrogen, Carlsbad, CA) as previously described [24] . Cells were incubated with DiD-LDL (10 mg/ml) at 37 C for 2 h. At the end of the incubation period, cells were harvested and washed three times with PBS containing 0.5% BSA before analysis by flow cytometry.
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