Pageruler

Samples of cultures containing similar number of cells were centrifuged, supernatants were removed, and the cell pellets stored at –80°C. Pellets were resuspended in 1× SDS Laemmli buffer (52 ), and lysed by heating at 98°C for 10 min. Proteins were resolved by Tricine-SDS PAGE (56 (link)) and then electroblotted to PVDF membranes. C.Csp231I protein bands were detected by chemiluminescence using the ECL-plus Western Blotting Detection System (GE Health Sciences) with 1:2000 dilution of rabbit anti-C.Csp231I polyclonal serum prepared according to standard protocols (57 ), and a 1:30 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. Protein bands were visualized either by autoradiography or by using 5-bromo-4-chloro-3-indolylphosphate (BCIP) as the alkaline phosphatase substrate and nitroblue tetrazolium (NBT) as the color development reagent. The prestained MW markers used were PageRuler (Fermentas).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE) was conducted according to [41 (link)]. Pre-stained standards (PageRuler; Fermentas, Ontario, Canada) were used to estimate the molecular weights of the sample proteins. The samples were stained with 0.25% Coomassie Brilliant Blue R-250 (w/v) and destained with a mixture of methanol/acetic acid/water (v/v/v; 4:1:5). After SDS-PAGE, the purified NAGase band was manually excised from the gel, prepared, and analyzed by LC-MS/MS mass spectrometry (XEVO-TQS mass spectrometer; Waters, San Diego, CA, USA) coupled with a UPLC chromatography system (Waters, San Diego, CA, USA) according to [42 (link)]. Raw data were converted to mzXML and automatically processed using Labkey Server v. 12, using theX!Tandem search algorithm [43 (link)].

Immunoblotting was carried out as previously described54 (link). Briefly, equal amounts of whole cell extracts (30 μg) or equal volumes of pull-down materials were analyzed by 10% SDS-PAGE under reducing conditions. The molecular weight of proteins was confirmed using prestained protein ladder (PageRuler, Fermentas). The R1(57) anti-APP C-terminal rabbit antibody55 (link), a kind gift of Dr. S. Efthimiopoulos (University of Athens, Greece) and the rabbit anti-AUF-1 antibody (07-260, Millipore) were used in a dilution 1:2000, the mouse anti-ELAVL and the rabbit anti-U2AF65 antibodies (3A2 and H300, respectively; all from Santa Cruz Biotechnology) in a dilution 1:1000 and the mouse anti-SAP97 (K64/15 UC Davis/NIH NeuroMab Facility and Antibodies Inc.) in a 1:50 dilution. Finally, the mouse anti-β-tubulin, the mouse anti-βΙΙΙ-tubulin (T5201 and T8660, respectively from Sigma) and the mouse anti-GAPDH-HRP conjugated (HRP-60004 from Proteintech) antibodies were used in a dilution 1:5000. All secondary HRP-conjugated antibodies (Santa Cruz Biotechnology) were used in a 1:5000 dilution. Each sample was tested in duplicate and samples obtained from three independent experiments were used for analysis. Densitometric analysis of immunoblotting images was performed using the image analysis software Image J (NIH, USA).

Equal amounts of denatured protein were subjected to 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), including molecular weight markers (PageRuler, #26617, Fermentas and PageRuler Plus, #266201, Fermentas) to estimate the molecular weight sizes of appearing bands, and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were washed in TBST buffer (TBS containing 0.1% Tween-20), and non-specific binding sites were blocked by immersing the membranes in blocking buffer containing 5% non-fat milk (70166, Sigma) in TBST buffer for 1 h on a shaker at room temperature or overnight at 4 °C. Membranes were first probed with the following primary antibodies: α-MMP2 (1:1000, sc-10736, Santa cruz), α-MMP3 (1:1000, ab52915, Abcam) and α-MMP14 (1:1000, ab3644, Abcam). After the first blotting, an anti-vinculin antibody (1:50000, V9131, Sigma) was used to control for protein loading. Next, a peroxidase-conjugated α-rabbit (111-035-144, Jackson ImmunoResearch) secondary antibody was used. Bound antibodies were visualized with the Pierce Enhanced Chemiluminescence (ECL) Plus Western Blotting Substrate detection system (32132, ThermoFisher) according to the manufacturer’s instructions.

Recombinant fungal strains were cultivated in 20 mL 2×MM medium containing 10% glucose in 125 mL Erlenmeyer flasks for 48 hr at 30°C and 200 rpm. Crude supernatant protein samples (15 mL) were separated using an 8% polyacrylamide separation gel (Laemmli, 1970 (link); Sambrook et al., 1989 ) at 100 V for 90 min. Proteins were visualised using the silver staining method (O'Connell and Stults, 1997 (link)). The prestained PageRuler^TM (Thermo Fischer Scientific) was used for size estimation of the recombinant endoglucanases.

Immunoblots were performed according to [11 (link)]. For each membrane, a housekeeping protein is depicted (HSP90, GAPDH, vinculin), which is always shown underneath the detected proteins of interest. Antibodies were: BCL-XL (#ab32370), GAPDH (#ab128915), WT1 (#ab89901) from Abcam, Cambridge, U.K.; RAF1 (#sc-133), HSP90 (#sc-13119), vinculin (#sc-736), ɣH2AX (#sc-101696) from Santa Cruz, Heidelberg, Germany; cleaved caspase-3 (#cs9661), p-Tyr202/Tyr204-ERK1/ERK2 (#cs9101), ERK1/ERK2 (#cs9102) from Cell Signaling, Leiden, Netherlands; p-Tyr694-STAT5 (#MA5-14973) from Thermo Fisher, Braunschweig, Germany; STAT5 (#610192) from BD Bioscience, Heidelberg, Germany; and PARP1 (#556362) from Pharmingen, Heidelberg, Germany. The protein ladders used were the prestained Scientific^TM PageRuler^TM (#26617) and the PageRuler^TM Plus (#26620) from Thermo Fisher, Braunschweig, Germany.