Immobilon p membrane

As described in Trostnikov et al. (2019) (link), for evaluation of the protein amount in motor neurons and the brains, approximately 30 thoraxes and 30 heads, respectively, of 3–5 days old adults of each genotype were dissected and homogenized in 8 M urea solution. Equal amounts of samples from the supernatants were preincubated with sample buffer (deionized water, 0.5 M Tris-HCl, glycerol, 10% SDS, 0.5% bromphenol blue, DTT) for 5 min at 95°C and separated in a 4–12% (w/v) acrylamide Bis/Tris SDS-PAGE gel using the vertical electrophoretic chamber Mini-Protean Tetra (Bio-Rad). Proteins were transferred from the gel to the PVDF membrane (Immobilon-P Membrane) using electroblotting (Mini Trans-Blot Modul, Bio-Rad), blocked in BlockPro blocking buffer (Visual Protein) and incubated with anti–GSK3 beta primary antibodies (1:300; ab18893, Abcam) for 1 h. Bound antibodies were detected with goat anti–rabbit secondary antibodies conjugated with alkaline phosphatase (1:20000; A3687, Sigma). Prior to visualization, the membranes were incubated in the alkaline CDP buffer for 5 min and then in the Immun-Star AP- Substrate (Bio-Rad) for 7 min. After scanning, the relative intensity quantification of each band was evaluated with Image Lab software (Bio-Rad). Three to four independent protein extractions (biological replicates) per sex per genotype were made.

Western blot analysis was conducted using the protocol established in the laboratory with some modifications (24 (link)). In brief, brain tissue was homogenized in 500-μl cell lysis solution containing 5% sodium dodecyl sulfate (SDS), 50 mM Tris–HCl (pH 7.4), and 1 mM ethylenediaminetetraacetic acid per 50–100 mg tissue. The homogenates were centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was collected. The concentration of the total brain protein was measured using the Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, MA, USA). A total of 30 µg of brain protein was separated on 7.5% SDS-PAGE gels at constant voltage (100 V) for 1 h, then transferred onto an Immobilon-P Membrane (polyvinylidene fluoride) for 2 h with constant voltage (100 V) at 4°C using Tank Transfer Systems (Mini Trans-Blot Cell, Bio-Rad Laboratories Inc., Hercules, CA, USA). After blocking with 10% skim milk for 1 h, the membrane was incubated with polyclonal rabbit antibody against NLGN2 (1:1,000) (Synaptic Systems, Goettingen, Germany) at 4°C overnight with shaking. Goat anti-rabbit IgG conjugated with alkaline phosphatase (1:7,500) (Promega, Madison, WI, USA, Wisconsin) was used as the second antibody. Immunoreaction was detected by ProtoBlot II AP system (Promega, Madison, WI, USA). Image J was used to measure the intensity of the signal.

Powdered tissue or collected cultured cells were resuspended in lysis buffer (20 mM Tris-HCL, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, pH7.5) with added protease and phosphatase inhibitor cocktails according to manufacturers instruction (Sigma). After lysis, lysates were cleared by centrifugation at 10 000g for 10 min at 4 °C. Protein concentrations of the supernatants were determined by D_c Protein assay (Bio-Rad). Proteins were diluted in Laemmli buffer with 1% 2-mercaptoethanol. Proteins (20 μg) were separated by SDS–polyacrylamide gel (10%) electrophoresis and transferred to Immobilon-P membrane using a semi-dry transfer apparatus (Biorad). Membranes were blocked for 1 h at room temperature and incubated overnight at 4 °C with the indicated antibody. Bound primary antibodies were detected using peroxidase-coupled secondary antibodies and enhanced chemiluminescence (Amersham). Relative quantification of band intensities was calculated by digitally photographing exposed films and using Genesnap and Genetools software (Syngene). Uncropped scans of blots are shown in Supplementary Fig. 2.

After the treatment of the indicated concentration of 10 μM oxaliplatin, complex 3 and complex 3a for 24 h, A549 and NCI-H1299 cancer cells were collected and lysed in lysis buffer (100 mM Tris-Cl, pH 6.8, 4% (m/v) sodium dodecylsulfonate, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin). Lysates were centrifuged at 12,000 g for 0.5 h at 4°C. The concentrations of total proteins were measured using the BCA assay method with Varioskan spectrofluorometer and spectrophotometer (Thermo, Waltham, MA) at 562 nm. Protein (20–100 μg) prepared from the indicated cells was loaded per lane and electrophoresed in 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) Immobilon-P membrane (Bio-Rad, USA) using a transblot apparatus (Bio-Rad, USA). The membranes were blocked with 5% (w/v) non-fat milk at 0.5 h at 37°C, followed by overnight incubation at 4°C with primary antibodies diluted in PBST. After washing with PBST, the membranes were incubated for 1 h with an IRDye´800 conjugated secondary antibody diluted 1:20000 in PBST, and the labeled proteins were detected with an Odyssey Scanning System (Li-COR., Lincoln, Nebraska, USA).

Dot blot analyses were carried out according to standard protocols, using Immobilon-P membrane and the BioDot SF apparatus (Bio-Rad, Munich/Germany). Thirty μl of the in vitro kinase reaction products described above or 20 μg of each EPIYA peptide were mixed in 1 mL of transfer buffer (192 mM glycine, 25 mM Tris-HCl, 20 % methanol, 0.1 % SDS, pH 8.3). Subsequently, the samples were spotted onto the Immobilon-P membranes (Merck Millipore, Darmstadt/Germany). After drying, the Dotblots were incubated with the various antibodies as described below for the Western blots.

Whole-cell protein extracts used for western blot analysis were collected as reported above. The proteins were separated on 6% polyacrylamide gel: the latter run was at 120 V for about 2 h, and the electrophoretic run was stopped when the 72 kDa blue marker left the gel. Then, it was transferred onto a polyvinylidene difluoride Immobilon P membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad), with the Turbo High precast protocol. The filters were blocked with 5% milk-TBST (50 mMTris pH 7.5, 150 mM NaCl, 0.1% Triton X-100) for 1 h at room temperature. The BRCA2 endogenous proteins were immunodetected using the Anti-BRCA2 (Ab-1) Mouse mAb (2B) (OP-95 Calbiochem, Merck KGaA, Darmstadt, Germany; 1:300). Vinculin (SC-7649, Santa Cruz Biotechnology, CA, USA; 1:1000) was used as a loading control. Anti-mouse and anti-goat IgG conjugated to horseradish peroxidase was used for the secondary antibodies (GE Healthcare, Chicago, Ill, USA). The bands were detected with ECL chemiluminescence methods (PerkinElmer, Waltham, MA, USA) and by exposure to the ChemiDoc MP System (Bio-Rad). For all Western Blot figures, the whole blot results are shown in Figures S7–S9).

LECs were lysed in buffer containing 20 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM NaF, 2 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche). Protein in the cell extract was quantified using protein quantification kit (Bio-Rad, Philadelphia, PA) and 10 μg total protein was run on Novex™ WedgeWell™ 4–20% Tris-Glycine Mini Gels (Invitrogen) and transferred to an Immobilon-P membrane (Bio-Rad). Membranes were probed with anti-p100/p52, NIK, phospho-IKKα/β (Ser176/180) (16A6), IκBα, TRAF2, TRAF3, and GAPDH antibodies. For co-IP assays, 500 μg total protein of cell extract was incubated with 1 μg of anti-mLTβR (5G11b) overnight, followed by 4 h incubation with 25 μL protein G Agarose beads (Sigma-Aldrich). The beads were then washed with lysis buffer and boiled in 2× Laemmli sample buffer (Bio-Rad). Uncropped immunoblotting images are included in Supplementary Fig 9.