Standard proteins

Separation of protein was performed by SDS-PAGE under reducing conditions and the transfer of proteins to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked with non-fat dry milk containing Tween® 20. Next, the membranes were incubated with the respective primary antibodies anti-PCNA (cat. no SAB2701819 Sigma-Aldrich; 1:500), anti-AR (cat. no. ab74272 Abcam, Cambridge, UK; 1:500), and anti-ZIP9 (cat.no. SAB3500599 Sigma-Aldrich; 1:500) at 4 °C overnight, followed by a horseradish peroxidase-conjugated secondary antibody (Vector Laboratories) at room temperature. Proteins were detected by chemiluminescence and documented with a ChemiDocTM XRS+ System (Bio-Rad Laboratories Hercules, CA, USA). The specificity of antibodies was assessed with the use of positive controls (rodent testes) (not shown). All immunoblots were stripped and re-probed with an anti-β-actin antibody (cat.no. A2228 Sigma-Aldrich; 1:300) as the loading control. The molecular weights of target proteins were estimated by reference to standard proteins (Sigma–Aldrich). To obtain quantitative results, immunoblots were analyzed densitometrically using the ImageLab software (Bio-Rad Laboratories) by an independent observer.

Nuclear extracts were prepared from the above 293 cells stably expressing Flag-USP36 as previously described (59 (link)). Briefly, cells were resuspended in hypotonic buffer A, homogenized and centrifuged as above. The resulting nuclear pellets were lysed in buffer C consisting of 20 mM HEPES (pH 7.9), 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.5 DTT, 25% glycerol with protease inhibitors. A total of 250 μl of the nuclear extracts were loaded onto a Superose 6 10/300 GL column (24 ml, GE) equilibrated with PBS (pH 7.2). The flow rate was 0.03 ml/min, and 56 fractions (300 μl each) were collected automatically. A calibration curve was obtained with standard proteins (Sigma-Aldrich) as run on the same system. Every two fractions were analyzed using IB with antibodies as indicated in the figure legends.

The molecular weight distribution of the 24 BSCH and a commercial hydrolyzed collagen (CHC) (obtained from Atlantic cod collagen) was estimated by gel filtration chromatography using an Agilent 1260 Infinity System (Agilent Technologies, Pittsburg, PA, USA) equipped with a Superdex Peptide 10/300 GL column (GE Healthcare, Uppsala, Sweden), with an isocratic elution using 0.1% Trifluoro-acetic acid (TFA) in 30% acetonitrile as the eluent. The samples of freeze-dried hydrolysates were dissolved in the eluent (10 mg/mL), filtered through a 0.45 µm filter, and ultrafiltered using a 10 kDa molecular weight cut-off Amicon Ultra Device. Aliquots of 10 µL were injected, eluted at 0.4 mL/min and monitored at 220 nm. The column was calibrated using the following standard proteins (Sigma, St. Louis, MO, USA): cytochrome C (12,400 Da), aprotinin (6500 Da), angiotensin II (1046 Da), leucine–enkephalin (555 Da), Val–Tyr–Val (379 Da) and Gly–Gln (221 Da).