Protein a g magnetic beads

To immunoprecipitate endogenous proteins, cell extracts were incubated with primary antibodies p14/ARF (AB11048, Abcam), RBX1 (ab133565, Abcam) or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G magnetic beads (Bimake) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting.

After being washed in PBS, the cells were lysed by being combined with lysis buffer for 30 min and then centrifuged at 4 °C Celsius for 10 min at 12,000 g. SDS‒PAGE was used to separate the cleared lysates. For Western blots, samples were transferred to PVDF membranes and identified using ECL detection reagents. For coimmunoprecipitation (CoIP), IP lysis buffer was used to lyse cells (Thermo Fisher Scientific, 87,788). First, the supernatants were incubated at 4 °C overnight with anti-Flag (Abmart, M20008) and anti-PD-L1 (ABMART, M033179) antibodies, followed by a 4 h incubation at 4 °C with Protein A/G Magnetic Beads (Bimake, B23202). The beads were rinsed five times with IP lysis buffer. Then, SDS-PAGE was used to separate the samples.

Total proteins or mitochondrial proteins were incubated with equal amounts of the anti-PKM2 antibody at 4 °C overnight after removing debris by centrifugation at 4 °C. Next, the immunocomplex was captured by adding Protein A + G magnetic beads (Bimake, Houston, TX, USA) according to the manufacturer’s protocols. The precipitate was washed five times with washing buffer. The samples were resuspended in 1× loading buffer and boiled for 10 minutes to dissociate the immunocomplex from the beads. Finally, the supernatant was collected by magnetic separation and subjected to Western blotting with anti-SIRT3 or anti-acetyl lysine antibodies (#ab21623, Abcam, Ltd. and then incubated with an anti-IgG heavy chain-specific secondary antibody (#A25222, Abbkine, Wuhan, China).

Protein-protein interactions were analyzed by co-immunoprecipitation (co-IP) as previously described (37 (link)). In brief, anti-E2F1 antibody and protein A/G magnetic beads (Bimake, Houston, TX, USA) were used to immunoprecipitate RB in cell lysates overnight at 4°C. Then the immunoprecipitated beats were collected and washed three times with lysis buffer. The proteins were separated by SDS-PAGE and analyzed by western blotting using anti-RB antibody.

We linked three labels (FLAG-DDX18, HA-Drosha, His-DGCR8) to the indicated proteins. The prepared cells transfected with FLAG-DDX18, HA-Drosha, and His-DGCR8 plasmids were collected for nuclear protein extraction followed by coimmunoprecipitation. A nuclear protein extraction kit (P0027, Beyotime) was used according to the manufacturer’s protocol. In brief, alternate vortexing and centrifugation combined with the extraction kit were used to separate the total proteins into nuclear and cytoplasmic proteins. At the same time as the cell nuclear proteins were prepared, protein A/G magnetic beads (B23201; Bimake, Shanghai, China) were preincubated on a spinning wheel at 4°C for 30–60 minutes and washed three times with PBS. The antibody complex was then suspended in the nuclear protein solution. After the protein solution was fully combined with the magnetic bead–antibody complex, the extraction buffer was washed three times. Magnetic separation was performed by heating. The immunoprecipitate was collected and western blotting was performed.

For western blotting, total cell proteins were separated by 10% SDS‒PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then incubated with primary antibody and horseradish peroxidase-conjugated secondary antibody. The proteins were detected using the ECL chemiluminescence system (NCM Biotech, Suzhou, China). Nuclear and cytosolic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer’s instructions (Beyotime, P0027, Shanghai, China). Lamin B and α-tubulin served as markers of the nuclear and cytoplasmic compartments. Image J software (Java 1.6.0_20, National Institutes of Health, USA) was used for the greyscale analysis of the scanned WB images. Validation of Fn and Fn-Dps antibodies specificity are shown in Fig. S16.
For Co-IP, to investigate the interaction between ATF3 and PD-L1 or Fn-Dps, the clarified supernatants were first incubated with anti-ATF3 antibody for 4 h at 4 °C. Protein A/G Magnetic Beads (Bimake, Shanghai, China) were then added, and after overnight incubation, the precipitates were washed five times with cell lysis buffer (Beyotime Biotechnology, Shanghai, China) and analyzed by western blotting. All antibody details are listed in Table S6.