Pierce immunoprecipitation kit

Calu-3 cells (5 × 10⁶) were inoculated with a multiplicity of infection (MOI) of 0.1 MERS-CoV at 37°C or under mock infection. At 24 hours post-infection (hpi), mock-infected and MERS-CoV-infected Calu-3 cells were harvested in lysis buffer for RIP without cross-linking using Pierce Immunoprecipitation Kit (Thermo Fisher Scientific, 26147) according to the manufacturer's protocol. To be more specific, the clear lysate of each sample was incubated with Protein A/G Plus Agarose beads conjugated with 10 μg mouse isotype-matched control IgG1 antibody or mouse anti-hnRNP C antibody (clone 4F4, IgG1) (sc-32308, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, respectively. Then beads were washed using washing buffer and the immunoprecipitated RNA-protein complexes were eluted for the detection of hnRNP C by Western blot, or were subjected to total RNA extraction and RT-qPCR for the detection of target circRNAs.

The co-IP assay was performed using the Pierce® immunoprecipitation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, AGS cells were lysed by ice-cold IP lysis/wash buffer and centrifuged at 13,000×g for 10 min to remove the debris. The supernatant was further immunoprecipitated with an anti-YAP1 antibody (1:50; Cell Signaling Technology). Rabbit IgG was used as a negative control. Precipitates were separated using SDS-PAGE and further analyzed by performing immunoblotting.

HEK293T cells were individually transfected or co-transfected with the expression plasmid for NF1-Flag, WT KRAS-GFP, or G12C KRAS-GFP. Cells were also simultaneously treated with either vehicle or 500 nM of AMG-510 for 24 h. Cells were harvested in IP Lysis/Wash Buffer (0.025 M tris-HCl, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, and 5% glycerol; pH 7.4 and 1× protease inhibitor) 24 h after transfection and treatment. Whole-cell lysates (500 μg) were precleared for 0.5 h using Control Agarose Resin slurry (Thermo Fisher Scientific). Immunoprecipitation was performed by first incubating 800 μl of HEK293T NF1-Flag precleared lysate with 200 μl of either WT KRAS-GFP, G12V KRAS-GFP or G12C KRAS-GFP precleared cell lysate. The final steps of the coimmunoprecipitation were performed using the Pierce Immunoprecipitation Kit (Thermo Fisher Scientific) with 10μg of immobilized anti-NF1 antibody (Santa Cruz Biotechnology, CA). A total of 500 μg of the cell lysate was added and incubated at room temperature under rotary agitation for 45 m. At the end of the incubation, the complexes were washed five times with lysis buffer. The western blot was probed with mouse monoclonal NF1 antibody (Santa Cruz Biotechnology) and mouse monoclonal RAS antibody (Thermo Fisher Scientific). All antibodies were diluted to 1/1000 in Licor intercept antibody dilutent (927–66003, Licor).