Ip lysis buffer

SNB-19 cells were grown to confluency and treated with EGF (50 ng/ml) for 5 min prior to lysis. To extract proteins, cells were washed with ice-cold PBS and lysed in IP lysis buffer (Thermo Fisher). Following a 10 min centrifugation at 10 000 g, the lysates were pre-cleared by incubation with 10 μl protein A/G-agarose beads (Santa Cruz) for 1 h at 4 °C with constant rotation. Lysates were then incubated with 2 μg Axl or EGFR IP antibodies for 2 h at room temperature, with constant rotation. Antibodies against GAPDH or Tensin2 (Santa Cruz) were used as species-aligned negative control IP antibodies. Following incubation, 20 μl of protein A/G-agarose beads was added to each sample and incubated further for 2 h. The beads were then separated by centrifugation at 8000 g for 1 min and washed once with ice-cold IP lysis buffer and thrice with PBS (Thermo Fisher Scientific). Finally, 4 × SDS-PAGE loading buffer was added to the beads and solubilised samples underwent SDS-PAGE and western blotting.

Immunoprecipitation was performed as previously described [22 (link)]. Briefly, cells were lysed with IP lysis buffer (Thermo Scientific, USA) containing the cocktail. Cell lysates were incubated with anti-Aurora-A antibody or PFKFB3 overnight at 4 °C, followed by immunoprecipitation with protein A + G agarose beads (Thermo Scientific, USA) for 2 h at 4 °C with shaking and washed 3 times with IP lysis buffer. The immunoprecipitate was eluted and analyzed by western blotting.

To confirm physical interaction between ORF3 and IKBKB, HEK293T cells were co-transfected with pCAGGS plasmids expressing ORF3-Myc and IKBKB-Flag or vice versa (ORF3-Flag and IKBKB-Myc for reverse co-immunoprecipitation). At 24 hpt, the cells were washed with cold phosphate buffer saline (PBS) and re-suspended in pre-cooled IP lysis buffer (Pierce^TM, Thermo Scientific) supplemented with protease inhibitor cocktail (Thermo Scientific). Cell lysates were cleared by centrifugation at 14,000× g for 5 min at 4 °C. Cleared lysates were then incubated with anti-Myc agarose (Pierce^TM, Thermo scientific) with gentle rocking overnight at 4 °C. Immunoprecipitates were washed with TBST buffer (25 mM Tris-HCl, 0.15 M NaCl, 0.05% tween 20, pH 7.2) and eluted in sodium dodecyl sulfate (SDS) sample loading buffer. The samples were subjected to SDS-PAGE and western blot.
For ORF3 and NEMO competition assay, each of plasmids expressing ORF3-Flag, IKBKB-Myc and HA-NEMO were transfected into HEK293T cells. At 24 hpt, cells were collected and lysed with pre-cooled IP lysis buffer (Pierce^TM, Thermo Scientific). An equal amount of lysate containing IKBKB-Myc and HA-NEMO and varied amounts of lysate with ORF3-Flag was combined and subjected to immunoprecipitation using anti-Myc bead as described above. Immunoprecipitates were eluted in sample buffer followed by SDS-PAGE and western blot.

Cells were harvested by scraping, and washed once with ice-cold phosphate-buffered saline (PBS) solution; after which, cells were incubated in IP lysis buffer (Life Technologies) supplemented with a protease inhibitor cocktail (Pierce). Protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). A 50 μL (1.5 mg) aliquot of Dynabeads® was transferred to a tube, which was then placed on a magnet to separate the beads from the solution, and the supernatant was removed. Next, Bcl-xl or Beclin-1 antibody in 200 μL of PBS containing Tween®-20 was added to the tube, which was then incubated with rotation for 2 h at 4 °C. After removing the supernatant, an equal volume of protein extract was added to the tube, which was then incubated with rotation overnight at 4 °C. Next, the Dynabeads®-Bcl-xL-protein or Dynabeads®-Belin1-protein complex was washed 3 times with 200 μL of washing buffer, the supernatant was removed, and 20 μL of elution buffer, 10 μL of premixed NuPAGE® LDS sample buffer, and NuPAGE® sample reducing agent were added to the tube, which was then heated at 70 °C for 10 min. Finally, the tube was placed on a magnet and the sample was loaded onto a gel.

Cells were lysed in immunoprecipitation (IP) lysis buffer (Life Technologies) supplemented with Mini protease inhibitor cocktail (Roche Diagnostics). Lysates were resolved by SDS/PAGE and probed with indicated antibodies (Abcam). The blots were subsequently stripped and re-probed with antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#ab-9485, Abcam) each time to ensure equal loading.