A9044

Tumor samples were processed as described earlier [54 (link)]. Preparation of cell lysates, SDS-PAGE and immunoblotting were performed as described elsewhere [55 (link)]. In brief, 40–80 µg protein per lane was transferred to PVDF membranes using a semi-dry transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked for 60–90 min with non-fat dry milk powder (5%, w/v) in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBS-T). For detection of Sig1R, membranes were incubated with primary antibodies in bovine serum albumin (BSA, 2% w/v) in TBS-T (PA5-30372, 1:500, Thermo Fisher Scientific (Waltham, MA, USA), (tumor samples), respectively, ab53852, 1:200, Abcam (Cambridge, UK) (cell lysates)), followed by incubation with peroxidase-conjugated secondary antibody (anti-rabbit IgG, A0545, 1:5000, Sigma-Aldrich, Steinheim, Germany). Proteins were visualized using Super Signal West Pico/Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and a CELVIN^®S Chemiluminescence Imaging system (Biostep, Burkhardtsdorf, Germany). For detection of loading control, membranes were stripped and further processed using mouse anti-β-actin antibody (A5316, 1:1000, Sigma-Aldrich) and anti-mouse IgG (A9044, 1:10,000, Sigma-Aldrich) as described elsewhere [54 (link)].

Proteins were extracted from PC-3 cells with ice-cold RIPA (Radio Immuno-Precipitation Assay) lysis buffer supplemented with protease inhibitor cocktail (11873580001, Roche), then homogenized in a dounce homogenizer for 1 h and centrifuged at 13,400 rcf at 4 °C for 10 min. The supernatants were collected and boiled with 6x sample buffer at 95 °C for 5 min. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using wet transfer or the semi-dry iBlot Transfer System (Invitrogen, Life Technologies). The membranes were blocked with 5% non-fat dry milk in PBS-T for 1 h, then incubated with either anti-CEBPB (Abcam 18F8, 1:1000 dilution), anti-AR (Cell Signaling 5153 S, 1:1000 dilution), anti-c-Myc (Santa Cruz N262,1:1000), anti-GAPDH (Santa Cruz Biotechnology, sc-322330 dilution) or anti-beta-Tubulin (Santa Cruz Biotechnology 3F3-G2, 1:8000 dilution) antibody in 1% non-fat dry milk in PBS-T overnight at 4 °C. Membranes were then incubated with secondary goat anti-mouse (Santa Cruz Biotechnology A9044, 1:10000) or goat anti-rabbit antibodies (Sigma-Aldrich A9169, 1:12,000) for 1 h at room temperature. Detection was achieved using the ECL Select detection reagent (Amersham, GE Health Care) with the ChemiDoc XRS + System (BioRad) (Supplementary Figs. 19 and 20).

We used the following antibodies in western blot analyses: mouse anti-Bcl-2 (B3170, Sigma), mouse anti-Bax (B8429, Sigma), mouse anti-RAD51 (SAB1406364, Sigma), mouse anti-β-actin (A5316, Sigma), and lgG-Peroxidase rabbit anti-mouse (A9044, Sigma).

The FgSR-Flag fusion construct was transferred into the wild-type strain PH-1, ΔFgSsk2, ΔFgPbs2, and ΔFgHog1, respectively. Each protein sample was extracted as described in Co-IP assays. The resulting protein samples were resolved on 8% SDS-polyacrylamide gels prepared with 25 μM Phos binding reagent acrylamide (APE × BIO, F4002) and 100 μM ZnCl₂. Gels were electrophoresed at 20 mA/gel for 3–5 h. Prior to protein transfer, gels were first equilibrated three times in transfer buffer containing 5 mM EDTA for 5 min and further equilibrated in transfer buffer without EDTA twice for 5 min. Protein from the Zn²⁺-phos-tag acrylamide gel to the PVDF membrane was transferred for 4–5 h at 100 V at 0 °C, and finally the membrane was analyzed by western blotting using an anti-Flag antibody (A9044, Sigma, St. Louis, MO; 1:10,000 dilution). The experiment was conducted independently three times.

Primary antibodies used were mouse-anti-NDPK-B (MC-412, Kamiya, Seattle, WA, USA, 1:1000), rabbit-anti-NDPK-A (sc-343, Santa Cruz, Heidelberg, Germany, 1:500; detects both NDPK-A and NDPK-B in mouse retinae), mouse-anti-Ang-2 (sc-74403, Santa Cruz, Heidelberg, Germany, 1:500), mouse-anti-O-GlcNAc (ab-2739, Abcam, Cambridge, UK, 1:1000), rabbit-anti-OGA (SAB4200267, Sigma, Munich, Germany, 1:1000), rabbit-anti-OGT (O-6264, Sigma, Munich, Germany, 1:1000), rabbit-anti-N1-phosphohistidine (MABS1330, Millipore, Darmstadt, Germany, 1:1000), mouse-anti-γ-tubulin (T6557, Sigma-Aldrich, Munich, Germany, 1:2000), rabbit-anti-GFAT (obtained in cooperation with Weigert, Tübingen), and sheep-anti-pGFAT (MRC-PPU s343c) for immunoblotting. The secondary antibodies used were rabbit anti-mouse peroxidase (A9044, Sigma-Aldrich, Munich, Germany, 1:20,000), goat-anti-rabbit peroxidase (A9169, Sigma-Aldrich, Munich, Germany, 1:40,000), and donkey-anti-sheep peroxidase (Sigma-Aldrich). qPCR primers were obtained from Applied Biosystems, ThermoFischer: GFAT Hs00899865_m1, OGA Hs00201970_m1, OGT Hs00269228_m1, and 18S Hs03003631_g1. Gelatin from porcine skin (48720, Fluka, Bucharest, Romania) was used as a 1% solution in PBS. Thiamet G (TMG; SML0244, Sigma-Aldrich, Germany) treatment was given at 10 µM for 24 h.