Peroxidase conjugated goat anti mouse secondary antibody

The plasmid pSGR-JFH1 encoding a sub-genomic replicon of JFH-1 strain was obtained from Dr T Wakita [27 (link)]. A BglII and an NsiI restriction sites were inserted between codons Pro419 and Leu420 of NS5A, and the coding sequence of enhanced green fluorescent protein (EGFP) was then inserted between these two sites. This position was previously shown to accept a GFP insertion in a sub-genomic replicon of the Con1 strain [28 (link)]. The plasmid was in vitro transcribed before electroporation into Huh-7 cells. Cells that express replicon were selected for using 500 μg/mL of geneticin during 15 days and cultured in a medium containing 250 μg/mL of geneticin. Huh-7 cells stably expressing replicon were seeded in 24-well plates and incubated with the different compounds for 24, 48 and 72 h. They were lysed in ice cold lysis buffer (Tris HCl 50mM, NaCl 100 mM EDTA 2 mM Triton-100 1% SDS 0.1%) containing protease inhibitors for 20 min. Cell lysates were collected and insoluble debris were removed by centrifugation. 20 μg of proteins were analyzed by western blotting using anti-GFP and anti-β tubulin antibodies. Peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch) was used for the revelation using ECL western blotting substrate (Thermo Fischer Scientific).

Huh-7 cells seeded in 12-well plates were inoculated with HCVcc for 2h. The inoculum was removed and replaced with culture medium. Forty-eight hours after infection, cells were lysed in ice cold PBS containing Triton-100 1% and protease inhibitors for 20 min. Cell lysates were collected and insoluble debris were removed by centrifugation. 20 μg of proteins were analyzed by western blotting using anti-E1 and anti-β tubulin antibodies. Peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch) was used for the revelation using ECL western blotting substrate (Thermo Fischer Scientific).

Slices of skeletal muscle biopsy of one LGMD2A patient and frozen skeletal muscle as normal control were dissolved in 100μl of saline solution and incubated at room temperature for 20 min before addition of EDTA 0.1M. Prior to sonication, the samples were resuspended in Laemmli 2X buffer (Sigma-Aldrich, Milwaukee, WI, USA), heated for 3 min at 95oC, and centrifuged. Protein extracts were run on 10% SDS-PAGE for 3hrs at 100V and electroblotted to PVDF membrane (Millipore, Milford, MA, USA). Blots were blocked with 5% low-fat milk powder and then incubated with monoclonal antibodies against Calpain-3 (Calp3c/12A2, diluted 1:50; Vector Laboratories, Burlingame, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab2302, diluted 1:300; Millipore) as internal control. Immunoreactive bands were visualized using a peroxidase-conjugated goat anti-mouse secondary antibody (Diluted 1:5000; Jackson Immunoresearch Laboratories, West Grove, PA, USA).