Tnfr2

Cells were collected and lysed in an appropriate precooled IP buffer. The cell lysate was centrifuged after being frozen for 30 min. Took 50 µl lysate supernatant as input. The rest was incubated with the indicated antibody against PGRN (Abcam, USA), TNFR2 (proteintech, USA), or IgG negative control (Santa, USA) on a rotating device at 4 ℃ for 2 h. Protein A/G Plus Agarose Beads (Santa, sc-2003) were washed 2 times with IP buffer and added to the cell lysate-antibody system on a rotating device overnight at 4 ℃. The next day, the agarose beads were washed 3 times and boiled. Samples were conducted by Western blot analysis. The total proteins were separated on SDS-PAGE and transferred onto the PVDF membrane (Merck Millipore, USA). After blocking with 5% milk for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4 °C. HRP-conjugated GAPDH Monoclonal antibody (Proteintech, HRP-60,004) was used as a loading control.

After various treatments as indicated, cancer cells were harvested, and the total protein was lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and then quantified by a BCA kit (Epizyme, Shanghai, China). Equal amounts of proteins were resolved on 10%-12.5% SDS-PAGE gels, followed by transferring the proteins to an Immobilon PVDF Membrane (Merck Millipore Ltd, Tullagreen, Ireland). PVDF membranes with proteins were blocked with 5% skim milk for 1 hour, incubated with primary antibodies overnight at 4°C, and then conjugated with secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 hour. Signals were visualized by ECL reagent (Epizyme, Shanghai, China) and photographed by Tanon 5200 visualizer (Tanon, Shanghai, China).
The primary antibodies used are as follows: p27, p21, Cyclin D1, PARP, cleaved PARP (c-PARP), cleaved caspase 3, cleaved caspase 9, cleaved caspase 7, cleaved caspase 8, Noxa, Bak, Bax, Bik, Bim, Bad, Puma, Bcl2, Bcl-xl, Mcl-1, XIAP, BID, TRAIL, DR4, DR5, CHOP, ATF4, eIF2α, and p-eIF2α were from Cell Signaling Technology (USA). Cyclin E, CDK2, CDK4, CDK6, Fas, DR3, c-Myc, and p53 were from Santa Cruz Biotechnology (USA). TNFR1 and TNFR2 were from Proteintech (USA). β-Actin was from HuaBio (China).

The protein samples of randomly 3 individual rat lung and liver tissues were loaded and resolved using 8, 10, or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then, the proteins were transferred to nitrocellulose membranes for 1 h, and at the solution of 5% skimmed milk, the membranes were blocked for an additional 1 h at 37°C. After that, they were incubated with the primary antibody overnight at 4°C: Cox-2, IL-1β, IL-6, TNFR-2, caspase-3, caspase-9, Bcl-2, Bax, p53, NF-κB p65, NF-κB p50, IK-α, and IKKβ (Proteintech, Wuhan, Hubei, China). Then, the membranes were washed in Tween-20 and Tris-buffered saline (T-TBS)/3 times and incubated at 37°C in secondary antibody (Proteintech, Wuhan, Hubei, China) for 1 h. Enhanced chemiluminescent (ECL) solution (Proteintech, Wuhan, Hubei, China) was used to visualize the membranes. And proteins were quantified by using Image Lab software (Bio-Rad, CA, USA) for 3 independent experiments. β-Actin was the corresponding expression of normalization [39 (link), 40 (link)].