A0168

Denaturing electrophoresis (SDS-PAGE) was performed on 30 μg of proteins employing a 4–20% gradient gel (#4561094, BioRad, Hercules, CA, USA). The following primary antibodies were used: anti-SRSF4 (#PA5-36366, ThermoFisher Scientific, Waltham, MA, USA), anti-phospho-mammalian target of rapamycin (mTOR, #5536S, Cell Signaling Technology, Danvers, MA, USA), anti-mTOR (#2983S, Cell Signaling Technology, Danvers, MA, USA), anti-clustered mitochondria homolog (CLUH, #A301-764A, Bethyl Laboratories, Montgomery, TX, USA), anti-OPA1 (#HPA036926, Merck, Darmstadt, Germany), anti-DRP1 (#ab184247, Abcam, Cambridge, UK), and anti-Actin (#MA1-140, ThermoFisher Scientific, Waltham, MA, USA). All primary antibodies were diluted following the manufacturer’s instructions in PBS plus 0.15% Tween 20 (PBSt; #11332465001, Roche, Basel, Switzerland). Specific secondary antibodies were employed (#A0168 and #SAB3700870, Merck, Darmstadt, Germany), all diluted 1:10,000 in PBSt. Bands were detected in the presence of an enhanced chemiluminescence substrate (ECL, #1705061, BioRad, Hercules, CA, USA) using a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC, Cambridge, UK). Band intensity was evaluated with Uvitec-1D 2015 software (UVITEC, Cambridge, UK). All bands of interest were normalized versus the actin signal detected on the same membrane.

ELISA was performed as described before in Bertoglio et al.³⁶ . In brief, 100 ng of antigen was immobilized per well on 96 well microtiter plates (MaxiSorp, Thermo Fisher Scientific) in PBS (pH 7.4) overnight at 4 °C. After coating, the wells were blocked with 2% MPBST for 1 h at RT, followed by three washing steps with H₂O + 0.05% Tween20. The tested IgG or scFv-Fc were added and incubated for ~ 1.5 h at RT. Following another washing step, bound IgG or scFv-Fc were detected by a goat anti-mouse IgG (Fc-specific) conjugated to horseradish peroxidase (A0168, Sigma, 1:42,000 dilution in 2% MPBST) or goat-anti-hIgG(Fc)-HRP (A0170, Sigma, 1: 70,000 dilution in 2% MPBST) depending on the Fc part used in the fusion construct. Visualization took place by addition of 100 µL/well tetramethylbenzidine (TMB) substrate (20 parts TMB solution A (30 mM Potassium citrate; 1% (w/v) Citric acid (pH 4.1)) and 1 part TMB solution B (10 mM TMB; 10% (v/v) Acetone; 90% (v/v) Ethanol; 80 mM H₂O₂ (30%))). After stopping the reaction by addition of 1 N H₂SO₄, absorbance at 450 nm with a 620 nm reference was measured in an ELISA plate reader (Epoch, BioTek). Data were analyzed by OriginPro 2019b (Lehre), using Hill function for EC₅₀ calculation.

Ovarian cancer cell lines (OAW28, OV-90) were treated with ATRA (1, 5 μM) for 6 days to 80% confluence in 75cm² flasks. Cells were dislodged using a cell scraper and cell pellet were resuspended in 200 μl of RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M sodium chloride, 50 mM Tris- HCL and 1 mM EDTA, pH 8.0 with protease inhibitor) spun at 7000 rpm for 10 min and stored at − 20 °C. Equal amounts of protein were electrophoresed and transferred to PVDF membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK) as described previously [6 (link)]. Proteins bands were detected with mouse monoclonal antibodies to annexin A2 (1/2000, Clone 5, 610069, BD Biosciences, North Ryde, NSW, Australia) or S100A10 (1/2000, Clone 148, 610070, BD Biosciences), anti-mouse IgG peroxidase-conjugated secondary antibody (1/4000, A0168, Sigma Aldrich), enhanced chemiluminescence (GE Healthcare), and ChemiDoc™ MP Imaging System with ImageLab™ software (Bio-Rad, Hercules, CA, USA) [8 (link)]. β-actin, used as a loading control was detected using a rabbit polyclonal antibody to β-actin (1/2000, ab8227, Abcam, Cambridge, MA, USA) and anti-rabbit IgG peroxidase-conjugated secondary antibody (1/4000, AP132P, Merck, Millipore, Bayswater, VIC, Australia).

Protein samples from CCM, tissue lysates, or TIF (10–80 μg) were used for western blot analysis. Protein samples were subjected to SDS-PAGE and transferred via a semi-dry system (Bio-Rad) to a polyvinylidene fluoride membrane (Amersham GE Healthcare, Buckinghamshire, UK). After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C. Subsequently, membranes were washed and probed with the corresponding secondary antibodies rabbit anti-goat POD (Sigma-Aldrich; A5420) or goat anti-mouse POD (Sigma-Aldrich; A0168) for 1 h at room temperature. After washing the membranes, they were developed using a Pierce West Pico/Femto Chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a Fusion SL Detection System (Vilber Lourmat, Eberhardzell, Germany).