Horseradish peroxidase conjugated anti rabbit antibody

Cells collected by scraping were disrupted by repeated passage through a 26-G syringe in 0.5 ml solution A (pH 7.4) containing 250 mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 μM aprotinin. After removal of cell debris by 14,000 rpm centrifugation at 4°C for 10 min, the supernatant was mixed 1:1 v/v with solution B containing 250 mM sucrose, 10 mM HEPES, and 1 mM MgCl₂. Following incubation at 4°C for 1 h, membrane fractions were centrifuged at 55,000 rpm at 4°C for 1 h and dissolved in a minimal volume of water. Protein (15 μg) was separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% skim milk in TBST at RT for 1 h, membranes were incubated overnight at 4°C with antibody against human Glut-1 (DAKO; 1: 1000) followed by 1 h incubation at RT with horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling; 1: 5000). Immune reactive proteins were detected and bands intensities were quantified as above.

Cell lysates were prepared, protein concentration determined, and fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. Membranes were immunostained with rabbit polyclonal anti-LDLR antibody (1∶2000) (Cayman Chemical, Cat. No. 10007665) for 16 h at 4°C and rabbit polyclonal IgG anti-GAPDH antibody (1∶1000) (Santa Cruz Biotechnology, Cat. No. SC-25778) for 1 h at room temperature and counterstained with a horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signalling, Cat. No: 7074s). The signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA). ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) was used to detect the signals, and Quantity One Basic 4.4.0 software (Bio-Rad) was used to quantify band intensities. The concentrations of the antibodies were optimized to achieve low background and a linear dose-dependent increase in signal intensity. The relative band intensities for the mature and precursor forms of LDLR protein expressed for the different constructs was calculated as the ratio between the LDLR 160 kDa or 130 kDa bands to that of GAPDH.

Exosomal proteins were extracted with a radioimmunoprecipitation assay buffer (Solarbio) supplemented with 1 mM phenylmethylsulfonyl fluoride (Solarbio) and incubated on ice for 20 min. Protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The protein lysates (30 μg) were subjected to 10% SDS-PAGE gel for separation and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% nonfat powdered milk in 1 × Tris-buffered saline tween (TBST) buffer for 1 h at room temperature and then incubated with primary antibodies against ALG-2-interacting protein X (ALIX) (Proteintech, Rosemont, IL, USA, 12422-1-AP, 1 : 1000), CD9 (Abcam, Cambridge, UK, ab92726, 1 : 2000), and heat shock protein 90 (HSP90) (Cell Signaling Technology, Danvers, MA, USA, #4877, 1 : 1000) overnight at 4°C. After washing with 1 × TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology, #7074S) for 1 h at room temperature. Bands were visualized using enhanced chemiluminescence reagent (Millipore) and detected by ChemiDoc system (Bio-Rad, Hercules, CA, USA).

HEK293 cells at a confluence of 5 × 10⁵ cells/well in 6-well culture plates (Sarstedt, Germany) were transfected with 1 µg cDNAs with Lipofectamine® LTX and PlusTM Reagent (Invitrogen). 24 h post-transfection, cells were washed and then incubated with fresh DMEM medium for an additional 24 h and then, media was recovered and secretion of PCSK9 was analysed by Western blot. For that purpose, proteins in the media were resolved by 8.5% Tris-Glycine SDS-PAGE. The gels were blotted onto Nitrocellulose membranes (Protran BA 83, Whatman™, GE Healthcare, Germany), blocked for 1 h in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk and immunoblotted with a rabbit polyclonal anti-human PCSK9 antibody (1:1000) (Cayman Chemical Company, USA, Cat.No: 10240) for 16 h at 4 °C. Then, counterstained with a horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signalling, Cat.No: 7074 s). The signal was developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA).

HNSC cell monolayers were lysed with 1× SDS sample buffer and dithiothreitol (DTT) reducing agent (AMS Biotechnology), and loaded onto a PAGEgel (Invitrogen). After electrophoresis, the proteins were blotted on nitrocellulose membrane. Immunodetection was performed by using rabbit anti-SOX5 and anti-Nor (NR4A3) polyclonal antibodies (Santa Cruz Biotechnology Inc.) and detected by using a horseradish peroxidase–conjugated anti-rabbit antibody (Cell Signaling Technology). The nitrocellulose membrane was then processed by using chemiluminescence-detection reagents (Thermo Scientific). The blots were stripped and reprobed by using anti-α-tubulin (Sigma, 1:1,000) to act as an internal loading-level standard. Western blot images were captured by using BioRad FluorS Imaging.