Pierce 660 nm assay

Cells were lysed in 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 10 mM EDTA, and 1% Triton X-100 supplemented with phosphatase and proteinase inhibitors (PhosSTOP/cOmplete, Roche). Total protein content was quantified with the Pierce 660 nm assay (Thermo Fisher Scientific) and equal amounts loaded into each lane. Proteins were separated on a 4%–10% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific) and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). See Supplemental Experimental Procedures for further details.

The protein concentrations of the co‐IP samples were estimated using the Pierce 660 nm assay supplemented with the ionic detergent compatibility reagent (Thermo Fisher Scientific, USA). A protein amount of 20 μg was subjected to proteolytic digestion using the filter‐aided sample preparation (FASP) protocol with 30 kDa Vivacon filters (Sartorius, Germany) as previously described (Wisniewski et al, 2009). Peptides were purified with self‐packed C18 stop and go extraction (STAGE) tips as previously described (Rappsilber et al, 2003).

PMNs were isolated as described previously [24 (link)]. Briefly, freshly drawn blood from healthy donors was collected in citrate tubes (ethical permit 2008/657 Lund University). Blood was layered on PolymorphPrep (Thermo Fisher Scientific, Waltham, MA, USA), and density gradient centrifugation was performed. Erythrocytes were lysed with endotoxin-free water and cells were finally resuspended in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA). After 4 h of incubation at 37 °C, cells were then mechanically activated by thermal shock. For this, samples were quickly switched between 45 °C and 0 °C to 45 °C, vortexed, and then spun for 20 min at 22,000× g to collect proteins. The samples were pooled from several donors and stored at −20 °C in aliquots until further usage. The overall protein content was determined using Pierce 660 nm assay (Thermo Fisher Scientific, Waltham, MA, USA). For SDS-PAGE, 2 µg were mixed (1:1) with reducing loading buffer (Thermo Fisher Scientific, Waltham, MA, USA), denatured at 95 °C for 10 min, and then loaded on a 10–20% Novex Tricine pre-cast gel (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresis was performed at 120 V for 90 min. The gel was stained using Coomassie Brilliant blue (Thermo Fisher Scientific, Waltham, MA, USA), and images were obtained using a Gel Doc Imager (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Neurons were collected in RIPA buffer with protease inhibitors (1 : 100, Fisher; Cat #: 78444), sonicated, and centrifuged (20 min, 14 000 g, 4°C). Protein in supernatant was quantified (Pierce 660 nm assay, Thermo Scientific, Waltham, MA, USA; Cat #: PI22663). Thirty‐five micrograms of protein were run on a 4–20% gradient polyacrylamide gel and transferred onto a 0.22 µm nitrocellulose membrane. Blots were blocked in 5% non‐fat dry milk (Bio‐Rad, Hercules, CA, USA, Cat #: 1706404) and incubated with primary antibody as follows: GluN2A (1 : 1000, Cell Signaling, Beverly, MA, USA, overnight, 4°C, RRID: AB_2112295), GluN2B (1 : 1000, Cell Signaling, overnight, 4°C, RRID: AB‐1264223), PSD‐95 (1 : 5000, Millipore, Burlington, MA, USA, overnight, 4°C, RRID: AB_10807979) or β‐actin HRP (1 : 5000, Cell Signaling, 1 h, (20℃), RRID: AB_1903890), followed by host‐specific secondary antibody for 1 h at 20℃ (1 : 2000 of goat anti‐rabbit HRP (Cell Signaling, RRID: AB_2099233) or goat anti‐mouse Alexa Fluor^® 647 (Probes, RRID: AB_2535804). Blots were imaged (Bio‐Rad Chemidoc) and band intensity was analyzed using Image Lab 6.0.1 (Bio‐Rad).

Testicular proteomes of young (n = 6) and old (n = 4) animals were analyzed. Then, 100 µL of lysis buffer (8 M of urea in 50 mM ammonium bicarbonate) was added to approximately 1 mg of testicular tissue. For lysis and homogenization, samples were sonicated using a cup resonator (Bandelin, Berlin, Germany) and further processed with QIAshredder (QIAGEN, Hilden, Germany) centrifugation devices (4 °C, 2500× g, 1 min). Protein concentration was determined with a Pierce 660 nm assay (Thermo Scientific, Waltham, MA, USA). Then, 20 µg of protein were reduced in 4 mM dithiothreitol/2 mM tris(2-carboxyethyl)phosphine and subsequently alkylated in 8 mM iodoacetamide. Proteins were digested in two steps: (i) Lys-C (1/100, enzyme/protein, FUJIFILM Wako, Neuss, Germany) for 4 h and (ii) trypsin (1/50 enzyme/protein, Promega) overnight at 37 °C.

Organic fractions from Trizol RNA extractions were stored at −20 °C until protein extraction was performed. To remove DNA from the organic fraction, 100% ethanol was added, and samples were centrifuged to pellet DNA. The phenol-ethanol supernatant containing the proteins was transferred to a fresh tube containing isopropanol and centrifuged at 4 °C to pellet the protein precipitate. The pellet was washed with 0.3 M guanidine thiocyanate in 95% ethanol and centrifuged at 4 °C. This wash step was repeated for a total of 3 times; 100% ethanol was added to the protein, which was then vortexed and incubated for an additional 20 minutes at RT. The protein was pelleted by centrifugation and air-dried at RT. Finally, the pellet was resuspended in urea lysis buffer (9 M Urea, 25 mM Tris-HCl, 1 mM EDTA, 1% SDS, 0.7 M β-mercaptoethanol). If necessary, the pellet was incubated at 50 °C for 10 to 20 minutes to successfully resuspend it in the urea buffer. Once resuspended, the sample was boiled and centrifuged, and the supernantant containing the protein was transferred to a new tube for quantification by the Pierce 660 nm assay with added ionic detergent compatibility reagent (ThermoFisher, Weltham, MA).

Unless specified otherwise, PSs (2.5 mg/mL polymer concentration) were incubated with 0.1, 1.0, or 10.0 mg/mL BSA, or with complete Dulbecco’s Modified Eagle Medium (CDMEM) supplemented with 10% FBS, at 37 °C, 80 rpm for 2 h. PS–protein complexes were isolated by ultracentrifugation at 25,000 × g, 4 °C for 30 min in a Beckman Coulter Optima MAX-XP ultracentrifuge operating under vacuum. The supernatant was removed, and PS–protein complexes were gently washed with sterile 1× PBS. This washing procedure was repeated twice to remove free protein. In the final step, PS–BSA complexes were gently resuspended in 1× PBS. The concentration of adsorbed proteins was determined using the Pierce 660 nm assay (Thermo Scientific) calibrated against a BSA concentration series (0, 0.125, 0.250, 0.5, 0.75, 1.0, 1.5, and 2.0 mg/mL). Absorbance of 660 nm light was measured using a SpectraMax M3 microplate reader (Molecular Devices).