Siah1

PLA was performed using Duolink PLA reagents (Sigma-Aldrich, DUO92008) according to the manufacturer's instructions. Briefly, 7 µm tissue cryosections were fixed in methanol as described above, followed by blocking with the provided blocking buffer and then incubated with primary antibodies against the two proteins of interest. The primary antibodies used for PLA analyses were: MAB21L4 (1:50, Biorbyt, orb159238), CacyBP (1:50, Novus Biologicals, H00027101-B01P), Envoplakin (1:50, Novus Biologicals, NBP2-37940), HA (1:50, BioLegend, 901501), Periplakin (1:50, LSBio, LS-C482731), CacyBP (1:50, Proteintech, 11745-1-AP), RET (1:50, NSJ Bioreagents, F53523), Ubiquitin (1:100, Abcam, ab7780), Siah1 (1:50, Genetex, GTX113268). After 2 washes, the tissues were incubated with the PLA probes anti-mouse MINUS (Sigma-Aldrich, DUO92004) and anti-rabbit PLUS (Sigma-Aldrich, DUO92002), followed by the ligation and amplification reactions. Tissues were then mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and imaged using a LSM 880 Airyscan Fast Live Cell inverted confocal microscope (Zeiss). Images of several z-stacks were acquired and the maximum intensity projection image was generated using the Fiji-ImageJ package.