Irdye 800cw goat anti mouse igg secondary antibody

HEK 293 T cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO₂ in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37 °C. Plasmid transfections were done with TransIT-LT1 (Mirus Bio) per the manufacturer’s instructions. Briefly, cell extracts were generated on ice in EBC buffer, 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP40, 1 mM DTT, and protease and phosphatase inhibitors tablets (Thermo Fisher Scientific). Extracted proteins were quantified using the PierceTM BCA Protein assay kit (Thermo Fisher). Proteins were separated by SDS acrylamide gel electrophoresis and transferred to IMMOBILON-FL 26 PVDF membrane (Millipore) probed with the indicated antibodies and visualized either by chemiluminescence (according to the manufacturer’s instructions) or using a LiCor Odyssey infrared imaging system. Western blot was conducted as described previously^{10 (link)}. Primary antibodies used for western blot are HA (Cat# 902302; 1:1000 dilution) from Biolegend and M2 FLAG (Cat# F1804; 1:1000 dilution) antibody from Sigma. Secondary antibodies used are IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (LiCor) and IRDye 680RD Goat anti-Rabbit IgG Secondary Antibody (LiCor). The study used a primary antibody against β-actin (Cat# A1978 from Sigma) for internal protein control.

In total, 6% and 10% polyacrylamide TBE gels were transferred onto a Biodyne B Nylon Membrane (Thermo Fisher Scientific, cat. # 77016) at 30 to 35 V for 1 h and 15 min on ice in 0.5× TBE using the Novex X-Cell II Blotting System (Invitrogen). After transfer, membranes were washed briefly in H₂O and fixed for 30 min at RT in Ca²⁺/Mg²⁺-free 1× PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde. Postfixation, membranes were washed 3× (10 min each) in Ca²⁺/Mg²⁺-free PBS containing 0.1% Triton X-100. Membranes were blocked overnight in Odyssey PBS Blocking Buffer (Li-Cor, cat. # 927-40000) and stained for 1 h at RT with Syn1 primary antibody (1:1000; BD Biosciences, cat. # 610787) and overnight at 4 °C with IRDye 800CW Goat anti-Mouse IgG secondary antibody (1:10,000; Li-Cor, cat. # 926-32210). Images were acquired using Li-Cor Odyssey CLx Imaging System. After DNA imaging, some gels were washed 1× (5 min) in MilliQ H₂O and stained in 15 to 20 ml of Coomassie G250 Safestain (Bio-Rad, cat. # 1610786) overnight at 4 °C on a rotator. Gels were washed 2× (1 h, then 2 h) in MilliQ H₂O. Images were acquired using a Li-Cor Odyssey CLx Imaging System.

E2+MPA treated organoid cell pellets were homogenized in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mg Na Deoxycholate with added protease (cOmplete, Sigma Alrich, St. Louis, MO, USA) and phosphatase (Phosphatase Inhibitor Cocktails 2 and 3, Sigma) inhibitors. Protein levels were assayed using the BCA kit (Thermo Fisher, Waltham, MA, USA), and 12 µg per lane was loaded onto a Protean mini gel (10 well, 10%, Bio Rad Hercules, CA, USA) run, and transferred to nitrocellulose membrane using the Turbo-Blot transfer system (BioRad) according to manufacturer’s directions. Membrane was blocked with 5% milk (Santa Cruz Biotech, Santa Cruz, CA, USA) in TBST (20 mM Tris, pH 7.4 (Lonza, Morrisville, NC, USA), 140 mM NaCl (Lonza), 1% TWEEN-20 (Sigma). PGR was detected using a mixture of IMA5 12,658 (Invitrogen, Waltham, MA, USA), hPRa7 (kindly provided by Dr. Dean Edwards), and mAb 1294 (Santa Cruz; sc 166169) each at 1:1000 in milk overnight at 4C. Bands were detected using IRDye 800 CW Goat anti-Mouse IgG Secondary Antibody (Licor, Lincoln, NE, USA) diluted 1:20,000 in 5% milk for 45 min, and images generated using a Licor Fc instrument with Image Studio software (Licor). β-ACTIN was similarly detected using Actin (I-19)-R (Santa Cruz; sc-1616-R) diluted 1:5000, and IRDye^® 680RD Goat anti-Rabbit IgG Secondary Antibody diluted 1:20,000.

Total cell lysates and biotinylated proteins were subjected to SDS-PAGE electrophoresis. The primary antibodies used include mouse anti-HA (901501, 1:5000, BioLegend), mouse anti-GAPDH (FD0063, 1:10,000, FuDeBio), mouse anti-Akt (sc-5298, 1:2000, Santa Cruz Biotechnology), rabbit anti-p-Akt (4060, 1:2000, Cell Signaling Technology). The secondary antibodies used were IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926–32210, LI-COR Biosciences), IRDye® 680RD Goat anti-Rabbit IgG Secondary Antibody (926–68071, LI-COR Biosciences). LY294002 (L832989, 10 μM, Macklin) was applied for 24 h to inhibit PI3K signaling.