Anti ldlr

Cell and liver protein samples were extracted and prepared as previously described [17 (link)]. Proteins were probed using the anti-HMGCR (1 : 1000; Novus), anti-ABCG5 (1 : 500; Proteintech), anti-NPC1L1 (1 : 1000, Novus), anti-PCSK9 (1 : 1000; Proteintech), and anti-LDLR (1 : 1000; Proteintech) and revealed using the horseradish peroxidase (HRP) conjugated secondary antibodies (1 : 2000, Cell Signaling Technology) and HRP-conjugated beta actin antibody (1 : 2000, Proteintech). The housekeeping protein beta-actin was used as an internal reference. Blots were visualized by enhanced chemiluminescence. Densitometry quantification of signals was conducted by ImageJ software.

Protein lysis from cells, exosomes and tissues were prepared and the protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA.). Protein Samples were separated by 10% or 12% SDS-PAGE gels and transferred to nitrocellulose filter membranes. The nitrocellulose filter membranes were blocked with 3% skim milk in tris buffered saline (TBS) containing 0.1% Tween-20 (TBST) at 4 °C overnight, and then incubated with primary antibodies followed by a horseradish peroxidase-conjugated secondary antibodies (washed with TBST three times before each operation, 5 min each time). Primary antibodies used were anti-LDLR (10785-1-AP, Proteintech), anti-GM130 (sc71166, Santa Cruz), anti-TSG101 (ab83, Abcam), anti-CD63 (ab134045, Abcam), anti-GAPDH (60004-1-lg, Proteintech), secondary antibodies used were anti-Rabbit (7074, CST) and anti-Mouse (7076, CST).

For immunoblotting, cells were washed twice with ice-cold PBS and lysed at 4°C in RIPA buffer (containing 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and complete protease inhibitor; pH 7.4). Lysates were centrifuged at 10,000 g for 10 minutes at 4°C, and protein concentrations in the supernatant were measured with a bicinchoninic acid assay kit (Thermo Fisher Scientific). Equal amount of proteins (20–30 μg) were resolved by 4–20% Bis-Tris gels (GeneScript) and transferred to PVDF membranes using iBlot^® 2 Dry Blotting System (Thermo Fisher Scientific). Blots were probed using previously described primary antibodies: anti-LDLR (1:1000; Proteintech), anti-(P)RR (1:1000), and anti-tubulin (1:5000; Proteintech), and HRP-conjugated goat anti-mouse or goat anti-rabbit antibodies (1:5000; Jackson ImmunoResearch) and detected by ECL. Abundance of target protein was quantified by measuring band intensity using Image J, and corrected for the band intensity of β-tubulin of the same sample.