Protein g sepharosetm 4 fast flow

Cell extracts were prepared basically as described previously (23 (link)). In brief, subconfluent cells were lysed with a solubilizing solution (25 mM Tris-HCl, pH7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 (TX-100)) containing protease and phosphatase inhibitor cocktails (Nakarai Tesque), and subsequently sonicated. After centrifugation at 20,000g at 4 °C for 10 min, the supernatants were used as cell extracts. To detect Trop-2 in lung and liver tissues harvested from tumor-bearing nude mice, lungs and livers were cut into small pieces, and after washing with PBS, tissue extracts were prepared as described above. Trop-2 in the cell extracts was immunoprecipitated using anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) or anti-HA-tag mAb-Magnetic Agarose (Medical and Biological Laboratories). Trop-2 in the tissue extracts was immunoprecipitated using goat anti-Trop-2 antibodies and protein G-SepharoseTM4 Fast Flow (GE Healthcare). The immunoprecipitates and cell extracts were subjected to SDS-PAGE, followed by immunoblotting. The target proteins were detected as bands by chemiluminescence, and the densities of the bands were determined with Image J software (National Institutes of Health).

Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [50 mM Tris (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 1× protease inhibitor (Roche) and PhosSTOP (Roche)]. Whole cell extracts were processed for immunoprecipitation with the following antibodies at 4 °C overnight. anti-SOS1 antibody (sc-10803, Santa Cruz Biotechnology, Inc.), anti-HER2 antibody (#2165, Cell Signaling Technology) and anti-Normal Rabbit IgG antibody (#2729, Cell Signaling Technology). Protein G Sepharose^TM 4 Fast Flow (17-0618-01, GE Healthcare) was added and an hour after incubation at 4 °C, the beads were washed three times in ice-cold wash buffer [20 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween 20]. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies: anti-HER2 (#2165, Cell Signaling Technology) and anti-SOS1 (sc-10803, Santa Cruz Biotechnology, Inc.). For secondary antibodies, HRP-conjugated anti-rabbit IgG (#7074, Cell Signaling Technology) was used. Blots were visualized using Chemi-Lumi One Super (nacalai tesque, Inc.) and ChemiDoc^TM XRS + Imager (Bio-Rad Laboratories, Inc.) according to the manufacturer’s recommendations.

To obtain polyclonal antibodies anti-LgRec1ALP1, a group of five BALB/c mice were immunized subcutaneously (s.c.) in the base of the tail (0.2 mL/animal) with 10 μg of LgRec1ALP1 in TBS buffer and emulsified in 0.2 mL of Montanide ISA50V. The animals were boosted i.d. 15, 30 and 45 days later with the same dose of antigen with an adjuvant. For the collection of the antiserum, the mice were euthanized in a CO₂ chamber, whole blood was collected by cardiac puncture and the serum obtained by centrifugation (4 °C, 10 min, 800 g). IgGs were purified by affinity chromatography using Protein G Sepharose^TM 4 Fast Flow (GE Healthcare, Little Chalfont, UK), following the manufacturer’s protocol. The concentration was determined in duplicate by the bicinchoninic acid method [82 (link)] using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) using the BSA (Sigma Chemicals, St. Louis, MO, USA) as the standard curve following the manufacturer’s protocol. Purified mice IgGs against the recombinant enhanced green fluorescent protein (EGFP) were used as a control.