Mouse anti ha tag monoclonal antibody

We refer to the experimental procedures described in the previously published literature for co-immunoprecipitation analysis [38 (link)]. First, the pCMV/GA₅₀/Myc plasmid and pCMV/SQOR/HA plasmid were co-transfected into 293 T cells according to the transfection method described in Section 2.2. After 24 h, cell lysates were collected using Pierce™ IP Lysis Buffer (Thermo Fisher Scientific). Next, supernatants were immunoprecipitated with anti-Myc-Tag antibody or normal immunoglobulin G from rabbits for 2 h (4 °C). Beads of Protein G-Sepharose were then added for 1 h. Finally, the co-immunoprecipitation complexes were washed and identified by Western blotting using mouse anti-HA-Tag monoclonal antibody (Cell Signaling Technology). In addition, we performed immunoprecipitation of cell lysates with mouse anti-HA-Tag monoclonal antibody and Western blotting with rabbit anti-Myc-tag monoclonal antibody. In another set of experiments, we co-transfected pCMV/SQOR/Myc plasmids and pCMV/GA50/HA plasmids into 293T cells and performed co-immunoprecipitation as described above.

293 and A549 cells were seeded at 1x10⁶ and 0.8x10⁶ cells per 35mm dish, respectively. Next day, the cells were infected for 1 h at a multiplicity of infection (MOI) of 10 with AdEmpty or AdFAST-HA. Following a 48 h incubation, whole cell lysates were collected using the cell lysis buffer as described by Lieber et al. [27 (link)]. Samples were separated by 15% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The resulting membrane was probed with a mouse anti-HA tag monoclonal antibody (1:1000, Cell Signaling #2367) and goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170–6516). To confirm Ad replication, the membrane was reprobed for Ad5 fibre (1:20 000 mouse anti-fibre monoclonal antibody, clone 4D2, Neomarkers). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit anti-α-tubulin antibody, AbCam #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170–6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific). Densitometry was performed using AlphaEaseFC (Alpha Innotech).

Collagen I (acid-soluble from rat tail) was from Sigma (Gillingham, UK). The antibodies and their sources were as follows: antibodies for immunofluorescence: mouse anti-HA-tag monoclonal antibody (dilution 1:200; Cell Signaling Technology), rabbit anti-calnexin polyclonal antibody (dilution 1:200, Santa Cruz), Alexa Fluor 568-goat anti-mouse IgG (dilution 1:200; Molecular Probes), Alexa Fluor 568-goat anti-rabbit IgG (dilution 1:200; Molecular Probes), Alexa Fluor 488-goat anti mouse IgG (dilution 1:200, Molecular Probes). Antibodies for Western blotting: goat anti-DDR2 from R & D Systems (Abingdon, UK); mouse anti-phosphotyrosine, clone 4G10, from Upstate Biotechnology (Lake Placid, NY); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences UK, Chalfont St Giles, UK); rabbit anti-goat Ig-horseradish peroxidase (Zymed Laboratories, San Francisco, CA).