Analyses of G3BP1 Cell Permeable Peptide Interactions

Ancillary Data:  Analyses of G3BP1 Cell Permeable Peptide Interactions 

These analyses indicate that the axon regeneration-promoting G3BP1 190-208 cell permeable peptide [CPP] does not bind to endogenous G3BP1 or G3BP2 in vivo (Ancillary Figure 1A-B) nor does it bind to recombinant G3BP1 in vitro(Ancillary Figure 1C).  These data are referenced in the above manuscript (doi: https://doi.org/10.1101/2024.06.07.597743).

Methods – 

To test in vivo CPP interactions, 30 µg of biotinylated G3BP1 190-208 or scramble 190-208 CPP  in 10 µl 1x PBS of was delivered by injection into rat sciatic nerve. Sciatic nerves were harvested 16 hours after injection and lysed in RIPA buffer with + 0.01% Rapigest (Waters, # 186001860) rather than SDS as detergent. Equal total protein amounts (approx. 500 µg) were added to each pull down with M-280 streptavidin dynabeads (Invitrogen, 11205D) and rotated at 4°C for 3 hours. Beads were magnetically separated and washed 3 times with RIPA + 0.01% Rapigest to isolate biotin-peptides and associated proteins. Bound complexes were eluted in 1x Laemmli sample buffer, boiled at 95°C for 5 minutes, and then processed for electrophoresis and immunoblotting to PVDF membrane. After blocking as described in the main manuscript, blots were incubated in anti-G3BP2 (1:1000; Invitrogen # PIPA5101815) or anti-G3BP1 (1:1000; Sigma # G6046) followed by HRP conjugated secondary antibody.  Blots were developed with chemiluminescent reagent.

To test for peptide interaction with recombinant G3BP1 protein, recombinant human G3BP1 (OriGene, # TP302692) alone or mixed in equimolar amounts of biotinylated G3BP1 190-208 or scramble 190-208 CPP in 1x PBS.  After 10 min at room 37°C, samples were incubated in formaldehyde (final concentration 1%) for crosslinking at room temperature for 15 min. Reactions were then quenched in 125 mM glycine then mixed with M-280 streptavidin dynabeads and rotated at 4°C for 3 hours. Beads were magnetically separated and washed 3 times with RIPA + SDS. Bound complexes were eluted in 1x Laemmli buffer and boiled at 95°C for 5 minutes followed by electrophoresis and immunoblotting as above.  Blots were probed with anti-G3BP1 (1:1000; Sigma # G6046) and processed as above for detection of protein bands.