Fig 2: (A) Protein sequence of p62 with protein domains underlaid in light blue boxes (Phox and Bem1p domain (PB1), Zinc finger-type (ZZ), TNF receptor-associated factor 6 (TRAF6), LC3-interacting region (LIR), protein sequence rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST), UBA. The six peptides identified after tryptic digestion by mass spectrometry are highlighted in red and blue. (B) Verification of the interaction of CB2 receptors with p62 by co-immunoprecipitation (Co-IP) of transiently overexpressed flag tagged CB2 receptors (FL-CB2) with endogenously expressed p62 in HEK293 cells. The precipitated p62 protein intensity directly correlated with the amount of transfected CB2-receptor plasmid (either 1 or 2 µg) that was not influenced by 10 min stimulation with the CB2 agonist HU-210. Representative result of n > 3.

Fig 3: (A) Flow chart of CaptiVate screening procedure. The bait CB2 was amplified and sub-cloned into the expression vector pN-TGSH (Dualsystems). The bait sequence was verified by sequencing. For constitutive expression, the bait was transfected into HEK293 Flp-In cells and selected for stable integration. Bait expression and solubility was monitored by immunoblotting using anti-HA antibodies. The expression level was compared to an internal standard and judged suitable for affinity purification and mass spectrometric analysis. (B) Background filtering of CB2 affinity purification: histogram of enrichment factors (CB2/control) of identifications to determine threshold of enrichment for specific interactors. Red area represents Gaussian distribution of unspecific interactors, yellow area is in the border and in the green area are the specific interactors (threshold as 10% of maximum peak height).

Fig 4: Functional Annotations were used to visualize protein–protein interaction network of 83 identified CB2 interactors by STRING. Illustrated interaction network was determined by text mining, experiments or databases with a minimum required interaction score of 0.400. In this view, the nodes represent proteins and the color saturation of the edges represents the confidence score of data support. CNR2 represents CB2 and SQSTM1 represents p62.

Fig 5: (A) Schematic illustration of Flag-p62 deletion constructs for ZZ, PEST, and UBA motifs that were used for following co-immunoprecipitation experiments to identify interaction region. (B) Stably expressing HA-CB2 HEK293 cells were transiently transfected with different Flag-p62-deletion constructs, respectively. Protein extracts were used for precipitation of Flag-p62 using Flag antibody. The expression levels in input samples for CB2 and p62 were comparable in all constructs. Immunoprecipitation result of Flag-p62 ?ZZ revealed a significantly reduced coprecipitation of CB2 indicating that this domain in p62 is involved in the interaction with CB2. Representative results of three independent experiments were presented.