Anti ldlr antibody

Stably Cas9-expressing HepG2 cell line (catalog no.: T3256), lentiviral sgRNA targeting human LDLR (catalog no.: 264181110204), and nonviral plasmids containing sgRNA targeting individual candidate genes were purchased from ABM Goods, Inc (Canada). Whole-genome lentiviral pooled CRISPR-Cas9 KO library (catalog no.: 73178-LV) was obtained from Addgene (Cambridge, MA). Alexa Fluor™ 568 carboxylic acid succinyl ester, Alexa Fluor™ 488-Human Transferrin conjugate (catalog no.: T13342), and DiI-human LDL conjugate (catalog no.: L3482) were obtained from Molecular Probes (Eugene, OR). Primer kits and Master Mix for RT-quantitative PCR (qPCR) were purchased from Applied Biosystems (Foster City, CA). Monensin, sodium salt (catalog no.: M5273), was purchased from Sigma-Aldrich (St. Louis, MO). Anti-LDLR antibody (catalog no.: 3839) was purchased from BioVision (Milpitas, CA), anti-TAGLN antibody (catalog no.: GT336) from Invitrogen (Carlsbad, CA), and anti-PCSK7 antibody (catalog no.: 19346S) from Cell Signaling Technology (Danvers, MA).

The liver isolated from a dead monkey was homogenized mechanically in radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris–HCl [pH 7.5], 150 mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L sodium fluoride, and 1 mmol/L sodium orthovanadate)^{59 (link)}. The lysates were centrifuged at 14,000 rpm for 15 min, and the supernatant was used for further experiments. Protein samples were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was then blocked for 1 h at room temperature in 5% BSA or 5% skimmed milk in Tris-buffered saline with Tween 20. The membrane was incubated with anti-LDLR antibody (1:1000 dilution) (Abcam) or anti-GAPDH antibody (1:2000 dilution) (Medical & Biological Laboratories, Tokyo, Japan) overnight in 5% skimmed milk at 4 °C, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (GE Healthcare, Piscataway, NJ, USA) for 1 h in 5% skimmed milk. The membrane was incubated with HRP substrate (Luminata Forte; Millipore Corp., Billerica, MA, USA) for 5 min and observed on a luminescent image analyzer LAS-4000 (Fujifilm Life Science, Tokyo, Japan).

Cell surface LDLR was detected by flow cytometry. Following treatment, LM3 cells were detached from the plates by scraping and washed with phosphate-buffered saline (PBS) containing 5% bovine serum albumin for 30 minutes at room temperature. Cells were then washed with PBS, incubated with an anti-LDLR antibody (Abcam) at room temperature for 1 hour, and then incubated with an Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Abcam) for 30 minutes at room temperature. Background fluorescence (control) was quantified using cells incubated with an isotype rabbit IgG antibody (Abcam) for 1 hour and Alexa Fluor 488-conjugated goat anti-rabbit IgG for 30 minutes. The cells were washed and resuspended in PBS, and then analyzed by flow cytometry using an FL1 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). The data were analyzed using the FlowJo software.
Dil-LDL (Biomedical Technologies Inc., USA) uptake was representative of total cellular cholesterol uptake. Following treatment of LM3 cells, the media was exchanged to serum-free and incubated with 10 ug/mL Dil-LDL at 37°C for 12 hours. The cells were detached with trypsin, washed, and the resuspended in PBS for flow cytometry analysis using an FL2 emission filter (FACScan, BD Biosciences, San Jose, CA, USA). Data were analyzed using the FlowJo software (CA, USA).

Cryosections were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.3% Triton X-100 for 5 min^{58 (link)}. Non-specific staining was reduced by blocking with 3–5% bovine serum albumin (BSA) for 1 h at room temperature. Samples were incubated overnight at 4 °C with anti-LDLR antibody (1:400 dilution) (Abcam, Cambridge, UK). The secondary fluorescent antibodies Alexa Fluor 488 and Alexa Fluor 555 (1:200–1:1000 dilution) (Thermo Fisher Scientific, Waltham, MA, USA) were applied for 1 h at room temperature in the dark. After washing samples with PBS, nuclei were stained with DAPI (1:200 dilution) (Dojindo, Kumamoto, Japan) for 5 min. The cells were viewed using a confocal microscope (FV1000-D; Olympus, Tokyo, Japan, or TCS SP8 X; Leica, Wetzlar, Germany). To visualize the cell membrane, cryosections were fixed in 4% paraformaldehyde, washed with PBS, and incubated with 5 μg/mL Wheat Germ Agglutinin, tetramethylrhodamine conjugate (Invitrogen) in PBS for 8 min. Then, the samples were permeabilized and stained with anti-LDLR antibody.