Myc tag

Isolation, quantification, SDS-PAGE and transfer protocols for total cellular proteins were performed as previously described [59 (link)]. Briefly, membranes were blocked with 5% nonfat milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature. After washing with 1× TBST, the membranes were incubated overnight at 4 °C with the following primary antibodies: RACK1 (Santa, sc-17754; CST, 5432s), β-catenin (CST, 8480s), cyclin B1 (Santa, sc245), cyclin D1 (CST, 2922s), cyclin E1 (CST, 20808s), HA tag (Abcam, ab137838), PSMD2 (Abcam, ab140675), GSK3β (CST,12456s), myc tag (Origene, TA300002), flag tag (Santa, sc-166355), β-actin (Sigma, A1978), and p-β-catenin-S33/S37T41 (CST, 9561s). After further washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and the protein bands were detected by chemiluminescence using an ECL kit (BIO-RAD, USA).

A PIK3C2B expression vector with a C-terminal Myc-Tag was purchased from Origene (cat. no. NM-002646). Primers with an adequate nucleotide mismatch were designed to engineer C1181F and H1208R amino acid exchanges into the plasmid. The implemented changes in the base triplicates were: C1181F: TGC>TTC / H1208R: CAC>CGC
To facilitate ensuing ligation, primers were additionally phosphorylated at the 5’-end.
Site-directed mutagenesis was carried out with the Phusion Site-Directed Mutagenesis kit (Thermo Scientific, cat. no. F541). Mutations were incorporated by following the indicated cycling conditions: initial denaturation (98°C, 10 min) was followed by 25 cycles of annealing (C1181F: 69°C, H1208R: 64,5°C, 20 sec) and extension (72°C, 5 min). PCR products were ligated (Promega, cat. no. M180S) and successful engineering was tested via Sanger sequencing.
An empty control plasmid was created by removing the PI3KC2B open reading frame via restriction digestion with NheI (Promega, cat. no. R650A) and MluI (Promega, cat. no. R638A). Plasmid fragments were separated in 1% agarose gel and purified (Promega, cat. no. A9281). Afterwards, 5’-overhangs were blunted (NEB, cat. no. M0210S) and ligated (Promega, cat. no. M180S). Plasmid constructs were cloned into E. Coli XL-1 Blue bacteria.

Uterine tissue was homogenized on ice in Tris-Triton-X100 tissue lysis buffer containing complete mini protease inhibitors (Roche, Basel, Switzerland). The tissue lysate was then incubated on ice for 20 min before the protein supernatant was obtained following centrifugation. Protein concentration was determined by the Bradford assay method (Biorad Laboratories, Hercules, CA). Protein (15 µg/lane) was resolved by a 4–15% gradient SDS-PAGE (Biorad Laboratories, Hercules, CA) before transfer to a polyvinylidene fluoride membrane (Millipore, Billerica, MA) [50] (link). Non-specific IgG binding was blocked with 5% milk in Tris-buffered saline (TBS) with 0.1% Tween. Immunoreactive bands were detected with the following antibodies: FLAG-tag (1∶1000 dilution, Sigma-Aldrich, St. Louis, MO), myc-tag (1∶1000 dilution,OriGene Technologies, Rockville, MD), SRC-2 (1∶2000 dilution, Bethyl Laboratories, Inc., Montgomery, TX), and β-actin (1∶10000 dilution, loading control; Sigma-Aldrich, St. Louis, MO). The immunoreactive bands were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL).