Anti mouse igg hrp a4416

Whole cell lysis (WCL) buffer was used to dissolve crushed tissue. WCL formulation was 10 mM Tris at pH 7.4, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 0.3% SDS, 1% Triton X-100 and the protease inhibitor cocktail [32 (link)]. The crushed tissue was dissolved in WCL buffer and transferred into an eppendorf tube and sonicated on ice. The lysate was then spun down at 16,000 × g for 3 min, and the resulting supernatant was used for sample preparation by mixing with 2x or 4x concentrated Laemmli buffer and boiled for 4 min. An aliquot of the lysate was used for Protein level assay using BCA method (Pierce). Proteins were resolved by SDS/PAGE and transferred to PVDF membranes (GE Healthcare). After probing with a specific primary antibody and horse radish peroxidise (HRP)-conjugated secondary antibody, the protein bands were detected with the ECL kit using X-ray medical film (Kodak). The antibodies used were anti-actin IgG (AC-15, Sigma), anti-pan CD44 IgG (2C5 BBA10, R&D Systems), anti-CD44v10 IgG (AB2082, Millipore), anti-mouse IgG-HRP (A4416, Sigma), anti-rabbit IgG-HRP (NA934VS, GE Healthcare).

To quantify infectious particle release, 1 × 10⁴ cells/well of Huh-7.5 cells were seeded onto 96-well plates. One day after plating, 6 replicate wells were infected with HCV infected supernatant, serially diluted and incubated for 72 h. The cells were then fixed with 100% methanol for 10 min, followed by 2 washes with PBS plus 0.1% Tween-20 (Sigma–Aldrich, St. Luis, MO, USA) (PBS-T). Endogenous peroxidases were blocked with 3% H₂O₂ in PBS-T for 5 min. The cells were subsequently incubated with 440 pg/mL of anti-NS5A 9E10 antibody (Cell Essentials, Boston, MA, USA) at room temperature for 1 h. Unbound antibodies were washed twice with PBS-T and the cells were incubated 1 h with anti-mouse IgG-HRP (A4416, Sigma Aldrich, St. Louis, MO, USA) at a 1:300 dilution. Peroxidase activity was detected after 2 X PBS-T washing and the addition of a carbazole substrate (0.32% w/v 3-amino-9-ethylcarbazole in N,N-dimethylformamide diluted in 5 mM acetic acid, 10 mM sodium acetate, pH 8.0 and 0.4% H₂O₂) for 10 min. Unbound substrate was washed twice with H₂O and stained cells were visualized under a microscope. Positive wells were counted and the tissue infective dose 50% (TCID50) was calculated using the Spearman and Kärber calculation [54 (link)].

Rabbit polyclonal anti-ADAMTS10 antibodies (pAb-867) were raised against a synthetic oligopeptide (SGHSKLPKRQRAC; in the second TSR domain). Other primary antibodies: mouse monoclonal antibodies against RECK (5B11D12) (Takahashi et al., 1998 (link)), FN (610078, BD), MT1-MMP (ab51074; Abcam) and α-tubulin (DM1A; Calbiochem/Merck Millipore, Burlington, USA). Secondary antibodies: anti-rabbit IgG-HRP (ab6721; Abcam), anti-mouse IgG-HRP (A4416; Sigma-Aldrich), anti-mouse IgG-CF488 (20018; Biotium, Fremont, USA), anti-mouse IgG-CF405M (20182, Biotium) and anti-rabbit IgG-CF647 (20282: Biotium). Antibody dilution: primary 1:1000 and secondary 1:20000 for immunoblot assays, primary 1:300 and secondary 1:1000 for immunofluorescence staining. Specificity of pAb-867 and 5B11D12 in immunofluorescence staining was assessed in MG-63 cells genetically manipulated to suppress (ADAMTS10) or inactivate (RECK) the expression of endogenous target genes (see Fig. S2).