Published April 29, 2024 | Version v1
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Developing nanobodies as allosteric molecular chaperones of glucocerebrosidase function

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OVERVIEW OF THE RAW DATA FILES
- DalMaso_et_al_Fig1A.xlsx: Raw data of the ELISA of 20 purified Nbs using GCase or N370S GCase coated on the bottom of the ELISA wells.
 
- DalMaso_et_al_Fig1C.xlsx: KD table of Nbs for GCase and N370S GCase.
 
- DalMaso_et_al_Fig 2B 2C: Results of GCase activity assay performed with 20 Nbs incubated with either velaglucerase or cellular lysates.
 
- DalMaso_et_al_Fig2E.xlsx: Thermal unfolding curves of GCase, obtained using a thermal shift assay, in absence or presence of Nbs.
 
- DalMaso_et_al_Fig4B 4C: Cytograms and raw data of GCase activity in live HEK293T cells transfected with ER- and lysosomal-targeted- Nbs.
 
- DalMaso_et_al_Fig4E 4F: Cytograms and raw data of lysosomal proteolytic activity evaluated in HEK293T cells transfected with ER- and lysosomal-targeted Nbs by DQBSA assay.
 
- DalMaso_et_al_Fig5A 5B: Western blotting of lysate of Nb-transfected HEK293T cells incubated with ENDO H and PNGase F.
 
- DalMaso_et_al_Fig5C 5D: Confocal images of HEK293T cells transfected with Nb1/Nb4/Nb9- flag-IRES-EGFP trageted to the ER stained with flag and calnexin for colocazation Nb-flag/ER-CALNEXIN. Pearson's coefficients calculated using Jacop plugin of ImageJ between FLAG and CALNEXIN.
 
- DalMaso_et_al_Fig5e 5f: Confocal images of HEK293T cells transfected with Nb1/Nb4/Nb9- flag-IRES-EGFP trageted to the ER stained with flag and LAMP2A for colocazation Nb-flag/lysosomes LAMP2A. Pearson's coefficients calculated using Jacop plugin of ImageJ between FLAG and LAMP2A.
 
 
- DalMaso_et_al_Fig6A 6B S15: Raw data of 4-MU assay performed with 20 Nbs incubated either with recombinant N370S or with tissue lysates from Gba-/- hN370S mice. Results of 4-MU assay perfromed on gut lysates from wild-type, heterozygous and knock-out mice. 
 
- DalMaso_et_al_Fig6C 6D S16E: Cytograms and raw data of GCase activity evaluated in live HEK GBA1 knockdown cells co-transfected with N370S GCase mutant and ER-Nbs and Lysosomal-Nbs. Cytograms of GCase activity in live wild-type, 11F GBA1 knock-down cell clone and 11F GBA1 knock-down overexpressing N370S mutant.
 
- DalMaso_et_al_FigS3.xlsx: Raw data of the ELISA of 20 purified Nbs using GCase or deglycosylated GCase coated on the bottom of the ELISA wells.
 
- DalMaso_et_al_FigS4.xlsx: Biolayer interferometry (BLI) sensorgrams data for the titration of GCase into a set of Nbs.
 
- DalMaso_et_al_FigS5.xlsx: Fluorescence polarisation raw data for Nb16-5TAMRA and GCase.
    
- DalMaso_et_al_FigS6.xlsx: Biolayer interferometry (BLI) sensorgrams data of GCase binding to immobilized Nbs (reverse set-up).
    
- DalMaso_et_al_FigS7.xlsx: Cathepsin L proteolysis assay raw data (western blot and quantification).
 
- DalMaso_et_al_FigS14.xlsx: Biolayer interferometry (BLI) sensorgrams data for the titration of GCase N370S into a set of Nbs.
 
- DalMaso_et_al_FigS10A S10B S11A S11B: western blotting of HEK293T cells transfected with Mock and ER- and lysosomal targeted Nbs to evaluate Nbs expression (FLAG) and their impact on ER (CALNEXIN) or lysosomal (LAMP1) compartments and on GCase expression (GCase)
 
- DalMaso_et_al_FigS10E: confocal images HEK293T cells overexpressing Mock sequence, Nb1, Nb4 and Nb9 stained with an anti-flag antibody, either targeted to the ER or to the lysosomes, with the relative EGFP fluorescence signal.
 
- DalMaso_et_al_FigS12A: cytograms and raw data of GCase activity in live HEK transfected with ER- and lysosomal Nbs and treated with GCase inhibitor CBE to evaluate the non-specific signal due to PFB-FDGlu off-target hydrolysis, or to other non-specific effects.
 
- DalMaso_et_al_Figs13: uncropped SDS-PAGE and blot of N370S GCase purified from 293T Freestyle cells 
 
- DalMaso_et_al_FigS16A S16B: western blotting of  6E and 11F GBA1 knock-down cell clones to evaluate GCase expression.
 
- DalMaso_et_al_FigS16C: cytograms of GCase activity in live wild-type and 6E and 11F GBA1 knock-down cell clones
 
- DalMaso_et_al_FigS16D: confocal images of  11F GBA1 knock down cell clone co-transfected with N370S GCase mutant and ER-targeted Nb4 (choosed as representative).
 
 
 

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DalMaso_et_al_Fig 4e 4f.zip

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Additional details

Funding

Michael J. Fox Foundation
GBA1 stabilization through nanobodies: a versatile tool to improve wild type and mutant enzyme function MJFF-17240
Michael J. Fox Foundation
GBA1 stabilization through nanobodies: a versatile tool to improve wild type and mutant enzyme function MJFF-020706
Research Foundation - Flanders
The Role of Parkinsonism genes in Synaptic Autophagy G031324N
Vrije Universiteit Brussel
Exploiting Peptides: Protein Binders, Modulators and Inhibitors for Imaging and Therapy SRP50
Vrije Universiteit Brussel
Exploiting Peptides as Protein Binders, Modulators, Inhibitors and Biomaterials for Imaging and Therapy SRP95

References