Image quant las 4000 luminescent analyzer

Crude cell lysates were prepared from cells using 100 µL RIPA buffer, 2 µL phenylmethylsulfonyl fluoride (1 mM), and 100 µL protease inhibitor cocktail (stock: 1 tablet/5 mL water). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA/MA). A total of 30 µg of protein was loaded in each well on an Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, USA/CA) and blotted onto a polyvinylidene fluoride (PVDF) membrane. Unspecific signal was blocked with 5% milk powder in TBST. The membranes were incubated overnight at 4 °C with primary antibodies: mouse α-PSMA (Abcam, 1:1000) or rabbit α-GAPDH (Cell Signaling, 1:2000). After washing, the secondary antibody horse α-mouse IgG HRP (Cell Signaling, 1:1000) or goat α-rabbit IgG HRP (Cell Signaling, 1:1000) was applied for 1 h at room temperature. The membrane was prepared for signal detection with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, USA/MA) and subsequently analyzed with an Image Quant LAS 4000 Luminescent Analyzer (GE Healthcare, USA/IL).

The recombinant protein extracted from yeast cells was separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) according to Laemmli (1970) (link) on 15% separation gels. For immunological detection, proteins were transferred from the SDS-gels to polyvinylidene fluoride (PVDF)-membranes (BioRad, Munich) by electro-blotting according to Jungblut and Seifert (1990) (link) using a ‘semi-dry’ blotting apparatus (Carboglass; Schleicher and Schüll). The membranes were incubated for 2 h in casein containing blocking solution and probed for the detection of the His-tag overnight at 4°C with, mouse anti-penta-His as primary antibody. Following incubation with the secondary antibody, i.e., goat anti mouse conjugated with horse radish peroxidase (Sigma, St. Louis, Missouri), the proteins were detected by chemo-luminescence following the application of SuperSignal West substrate (Thermo Fisher Scientific) in an ImageQuant LAS-4000 Luminescent Analyzer (GE Healthcare).

Human PSMA/isoform cDNA was amplified with the following PCR primer pairs (0.5 µM): PSMA: 5‘-TGCAGGGCTGATAAGCCAGGCATT-3‘ and 5’-TGGGATTGAATTTGCTTTGCAAGCTG-3’; PSM’: 5’-TGGACCCCAGGGTGGTTTAT-3’ and 5’-GCATCCCAGCTTGCAAACAA-3’; PSM-C: 5’-GGTACTGATTTGCAGACTTGATCC-3’ and 5’-GCTTTGGAGTAATGTTAGTAGCTTCAT-3’;PSM-D: 5’-CCAGGCAGGTTAAAAGCCAA-3’ and 5’-CCTCTCTGCCAGACACCCAG-3’PSM-E: 5’-GAATCTCCTTCACGAAACCG-3’ and 5’-ATAAACCACCCCAGCCTCTC-3’; PSMΔ6: 5’-AATTGGAACGGGACATGAA-3’ and 5’- CCTCTGCAATTCCACGCC-3’; PSMΔ18: 5’- GAAACAAACAAATTCAGCGG-3‘ and 5’-AGAGCATCATAAATTCCTGG-3’; human β-actin: 5’-AGAGCTACGAGCTGCCTGAC-3’ and 5’-CACCTTCACCGTTCCAGTTT-3’. Samples were diluted with 6 × loading dye and loaded on a 1% agarose/ethidiumbromide gel. A voltage of 100 V was applied for 1–2 h. Afterwards, the signal was detected with an ImageQuant LAS 4000 Luminescent Analyzer (GE Healthcare, USA/IL).