Dithiothreitol (dtt)

Peptide samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions using 4–12% Invitrogen NuPAGE Novex Bis-Tris precast gels (Thermo Fisher Scientific, Loughborough, UK) and the Invitrogen XCell SureLock Mini-Cell (Thermo Fisher Scientific, Loughborough, UK). An aliquot of 10 μL peptide samples was incubated with 4 μL NuPAGE lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) and 2 μL of 500 nM dithiothreitol (DTT) (Fluorochem, Hadfield, UK) for ten minutes at 95 °C and 15 μL of sample mixture loaded per lane and 5 μL of SeeBlue Plus2 pre-stained protein standard (Thermo Fisher Scientific, Loughborough, UK) loaded in at least one lane. The samples were run using 1X 2-morpholinoethanesulfonic acid (MES)-SDS running buffer (Thermo Fisher Scientific, Loughborough, UK). Electrophoresis was performed under a constant current of 125 mA for 40–60 min. The gel was stained using Biosafe Coomassie Brilliant Blue G-250 stain (Biorad, Watford, UK) for 60 min, washed and destained in water overnight and imaged using a Syngene G:box and GeneSnap software (Syngene, Cambridge, UK).

Hyaluronic acid (HA) with molecular weight 0.5–0.75 MDa was purchased from Contipro. Cystamine dihydrochloride (Cys, Sigma Aldrich, St. Louis, MO, USA), 3,3’-dithiobis(propanoic dihydrazide) (Hyd, FisherScientific, USA) and dithiothreitol (DTT, Fluorochem, Hadfield, United Kingdom) were employed for HA modification with thiol groups. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Fluorochem, Had- field, United Kingdom) and 1-Hydroxybenzotriazole hydrate (HOBt, Sigma Aldrich, St. Louis, MO, USA) were used as coupling reagents. Spectra/Por membranes (MWCO: 10 kDa) were employed for purification by dialysis. Phosphate-buffered saline (PBS, Sigma Aldrich P4417, St. Louis, MO, USA) was used as solution medium to form the hydrogels. 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB Ellman’s reagent, Sigma Aldrich, St. Louis, MO, USA), Ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich, St. Louis, MO, USA) and Cysteina-HCl (Thermo fisher Scientific, USA) were used for the determination of thiol groups.

PrestoBlue^® Cell Viability Reagent, Mem-PER™ Plus Membrane Protein Extraction Kit, and all reagents for cell culture were purchased from Life Technologies (Carlsbad, CA, USA). Bradford Protein Assay and Clarity™ Western ECL Substrate were obtained from Bio-Rad (Hercules, CA, USA). Protease inhibitor cocktail and lovastatin were purchased from Sigma (Saint Louis, MO, USA). Primary antibodies against Rab11A and Rap1A/Rap1B were obtained from Abcam (Cambridge, UK) and primary antibodies against β-actin along with secondary HRP-linked antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Iodoacetamide and WaLP were purchased from Sigma (Saint Louis, MO, USA), DTT, CuSO₄, TCEP from Fluorochem (Derbyshire, United Kingdom) and THPTA, biotin-PEG₃-azide from Click Chemistry Tools (Scottsdale, AZ, USA). Sequencing grade chymotrypsin was purchased from Promega (Madison, WI, USA).