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5 protocols using goat anti rabbit immunoglobulin g igg

1

Quantification of Gag.MS2 Protein Levels

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The content of Gag.MS2 precursor proteins in Gag.MS2 supernatants was analyzed by an immunoblot assay. In brief, 15 μL of each supernatant were mixed with 15 μL of Laemmli buffer and denatured for 5 min at 95°C. Next, 20 μL of each sample was separated by an SDS-PAGE (12.5% separation gel) and transferred to a nitrocellulose membrane (GE Healthcare Life Science, Solingen, Germany). Successful blotting and equal sample loading were controlled by staining the membranes with Ponceau S solution (40% methanol, 15% acidic acid, and 2.5 g/L Ponceau S). Afterward, the membranes were probed with a polyclonal rabbit-anti-enterobacteria phage MS2 coat protein antibody (1:10,000; Merck) in Tris-buffered saline with 0.05% (v/v) Tween and 3% milk powder (both Roth, Karlsruhe, Germany). As a secondary antibody, a goat-anti-rabbit immunoglobulin G (IgG) (1:2,000; Santa Cruz Biotechnology, Heidelberg, Germany) conjugated with horseradish peroxidase was used. Final visualization and quantification were carried out by treating the membrane with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and the FUSION FX imaging system (Vilber Lourmat, Eberhardzell, Germany), respectively.
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2

Immunohistochemical Analysis of Lubricin, RSPOs, and LGRs

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Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Immunohistochemistry
Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Biotechnology, USA), RSPO1 (Sigma-Aldrich, USA),
RSPO2 (Sigma-Aldrich, USA), LGR5 (Sigma-Aldrich,
USA), LGR6 (Abcam, England) and β-catenin (Santa
Cruz Biotechnology, USA) of 1:500 dilutions at 4˚C.
The slides were washed thrice with phosphate-buffered
saline (PBS, Wel Gene, Korea) and goat anti-rabbit
immunoglobulin G (IgG, Santa Cruz Biotechnology,
USA) was treated at room temperature for 30 minutes.
Western blot bands were developed for visualization with
3, 3′-diaminobenzidine as the chromogen. Each section
was photographed at ×10 magnification by a Zeiss Axio
Cam digital camera.
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3

Unfolded Protein Response Pathway Analysis

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Protein extracts were separated via 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membrane. After blocking membranes with 5% skim milk for 1 h, glucose-regulated protein 78 (GRP78) primary antibodies against PrPC, protein kinase-like endoplasmic reticulum kinase (PERK), phospho-PERK (p-PERK), inositol-requiring enzyme 1α (IRE1α), p-IRE1α, activating transcription factor 4 (ATF4), and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) were incubated overnight. After washing with PBS, the membranes were incubated with goat anti-rabbit immunoglobulin g (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, England, UK).
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4

Notch Pathway Activation in GSCs

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After 48 hours of infection, GSCs were collected and lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). A Bicinchoninic acid kit (Pierce, USA) was used to detect the protein concentration in the supernatant. Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% skim milk and probed with rabbit antibodies against jagged canonical Notch ligand 1 (JAG1; ab7771, 1:500), NOTCH1 (ab8925, 1:500), Presenilin‐1 (PS1; ab76083, 1:5000), HES family bHLH transcription factor 1 (HES1; ab71559, 1:1000) or GAPDH (ab9485, 1:2500, internal reference). All of aforementioned antibodies were obtained from Abcam (Cambridge, UK). The membrane was then re‐probed with goat anti‐rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) conjugated by horseradish peroxidase (HRP) for 1 hour. Enhanced chemiluminescence (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to develop the membrane, which was visualized in a Bio‐Rad ChemiDoc™ image analysis system. Protein bands were analysed by ImageJ2x software.
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5

Immunohistochemical Localization of Lubricin in Osteoarthritic Femoral Heads

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Random samples from femoral heads of various OA stages were immunostained with the two-step immunohistochemistry method as instructed by the manufacturer. In brief, deparaffinized sections were incubated overnight with rabbit polyclonal antibody against lubricin (sc-98454, Santa Cruz Biotechnology INC., CA, USA), 1∶200 dilution, at 4°C. The slides were washed three times in PBS followed by 30 min incubation at room temperature with goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology INC., CA, USA) and visualized with DAB chromagen. Stained sections were photographed with a Ziess AxioCam digital camera, at 20× magnification with constant illumination intensity (Figure 2).
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