Veriblot for ip detection reagent hrp

For coimmunoprecipitation experiments, cells were lysed in 1% digitonin for 30 minutes on ice. The lysates were incubated with primary antibody for 1–4 hours, followed by addition of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, catalog: sc-2003) overnight at 4°C. After 4 washes in 0.2% digitonin, samples were eluted in SDS sample buffer with 50 mM DTT for 10 minutes at 70°C, separated by SDS-PAGE, and immunoblotted. VeriBlot for IP Detection Reagent (HRP) (Abcam, catalog: ab131366) was used for immunoblotting.

Co-IP assays were performed as described previously^{76 (link)}. Antibodies were used for immunoprecipitation: MLKL (#GTX107538A, GeneTex), Skp2 (#2652, Cell Signaling Technology) or normal Rabbit IgG (NI01, Calbiochem). Immunocomplexes were resolved by SDS-PAGE and co-immunoprecipitated proteins were detected using Skp2 (#2652, Cell Signaling Technology), and MLKL (#66675-1-Ig, Proteintech), respectively. The VeriBlot for IP Detection Reagent (HRP) (#ab131366, Abcam) was used to avoid interference from denatured IgG heavy and light chains.

Immunoprecipitated protein (the entire immunoprecipitate from an independent IP experiment to that performed for mass spectrometry) was resuspended in 35 μl of 2 × loading buffer without bromophenol blue. Samples were boiled at 95 °C for 5 min and centrifuged at 18,000×g for 3 min at RT and bromophenol blue then added to the supernatants. 35 μl sample was electrophoresed for Western blot analysis, performed using rabbit anti-Importin beta (1:5000, Abcam ab45938), rabbit anti-CRM1 (H-300) (1:1000, Santa Cruz Biotechnology, sc-5595), rabbit anti-Kpnα2 (1:2500, Abcam ab97580), mouse anti-Ran (1:500, Sigma-Aldrich R4777), rabbit anti-CCAR1 (1:1000, Novus Biologicals NB500-186), rabbit anti-FUBP1 (1:500, Novus Biologicals NBP2-16543) and mouse anti-GAPDH (0411) (1:10,000, Santa Cruz Biotechnology, sc-47724). For all IP-WB analysis, Abcam Veriblot for IP Detection Reagent (HRP) (ab131366) was used as a secondary antibody with a dilution of 1:2500 in 5% milk in TBST. For analysis of whole cell lysates, 30 µg protein was loaded onto SDS-PAGE gels and blots probed using the same antibodies mentioned above. Lumiglo (KPL) was used as the chemiluminescent substrate for Western blot detection. For all Western blot analyses, membranes were cut prior to hybridisation with antibodies. Images of the original blots can be seen in the supplementary material (Supplementary fig. S11–S17).

Immunoprecipitation and co-immunoprecipitation were performed using a Pierce Classic Magnetic IP/Co-IP Kit (88,804, Thermo Fisher Scientific). Cells were lysed with cold lysis buffer and supernatant was collected after centrifugation at 13,000 g for 10 min. Approximately 1000 μg protein was incubated with specific IP antibody (1:50) at 4 °C on a rotating platform overnight. Pierce Protein A/G Magnetic Beads (25 μL) were added to the antigen sample/antibody mixture and incubated at room temperature for 1 h. After washing, the target antigen–antibody complex was eluted with 100 μL of Elution Buffer and 10 μL of Neutralization Buffer, followed by Western blotting analysis or mass spectrometry analysis at BGI (BGI, Shenzhen). VeriBlot for IP Detection Reagent (HRP) (ab131366, Abcam) was used to avoid the detection of heavy and light chains.