Sample preparation for PALM imaging

The homotrimeric SNAP-Flag-TNC-TNFα was labeled with a twofold excess of Alexa Fluor 647 benzylguanine (New England Biolabs) in PBS (note that the homotrimer contains three SNAP subunits, which ensures efficient labeling of the homotrimer with at least one fluorophore). For cell seeding, eight-well flexiPERM reusable slides (Sarstedt) mounted on glass slides (Thermo Fisher Scientific) were washed in isopropyl alcohol (Merck) for 15 min and plasma-cleaned in N₂ for 15 min (Diener Electronic) to reduce background. The cleaned slides were coated for 90 min with poly-l-lysine (PLL) (0.8 mg/ml; Merck) coupled to polyethylene glycol (PEG) (Rapp Polymere) and a peptide containing the RGD motif (PLL-PEG-RGD) to facilitate cell adhesion. For TNFR1-mEos2 SMLM, 1.5 × 10⁴ TNFR1/2^−/− MEFs stably reconstituted with TNFR1-mEos2 WT, TNFR1 K32A, or TNFR1 N66F were seeded in 300 μl per well of serum-free RPMI 1640 medium (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), penicillin (100 U/ml; Thermo Fisher Scientific), streptomycin (100 μg/ml; Thermo Fisher Scientific), and gentamicin (50 μg/ml; Thermo Fisher Scientific). After 24 hours of starvation, seeded cells were induced with 2.4 nM SNAP-Flag-TNC-TNFα (100 ng/ml) labeled with Alexa Fluor 647 in ice-cold serum-free medium at 4°C for 30 min. Cells were washed three times with 300 μl of 400 mM sucrose (Sigma-Aldrich); dissolved in sterile-filtered PBS (Thermo Fisher Scientific); and fixed in 300 μl of 4% formaldehyde (Thermo Fisher Scientific), 0.1% glutaraldehyde (Merck), and 400 mM sucrose in sterile-filtered PBS. After fixation, cells were washed three times with sterile-filtered PBS.