Ap21967

B2B1 cells were seeded on tissue culture plastic (6-well to 15-cm plates) at 12,500 cells/cm² in assay medium + 2% matrigel + 5 ng/ml EGF (Peprotech, AF-100-15) and refed every 4 days. On Day 6, cultures were spiked with 0.5 µM AP21967 (Takara, 635057) or 0.1% ethanol vehicle for 24 h before cell lysis and biochemical analysis.

Cells were washed twice with PBS and seeded at 1.0 × 10⁵ cells/ml into 24-well plates with or without various concentrations of AP21967 (Takara Bio). Three days later, cells were mixed with Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA) and 1 μg/ml propidium iodide (Sigma-Aldrich) and subjected to flow cytometry (FACSCalibur; BD Biosciences, Lexington, KY) to measure viable cell density. The data were analyzed using FlowJo software (BD Biosciences).

For B2B1 cells and human-rat clones, cells were plated at 5000 cells per well on growth factor-reduced matrigel (Corning, 354230) in 8-well chamber slides (Falcon, 354108) and re-fed every 4 days according to published methods^{63 (link)}. On Day 6, cultures were spiked with 0.5 µM AP21967 (Takara, 635057) or 0.1% ethanol vehicle, and these perturbations were retained in the culture medium during subsequent refeeds. For TM15c6 cells, cells were plated at 5000 cells per well on growth factor-reduced matrigel (Corning, 354230) in growth medium (DMEM medium (Gibco) plus 5% fetal bovine serum, 5 µg/ml insulin, 1 µg/ml hydrocortisone, 5 ng/ml EGF, 35 µg/ml bovine pituitary extract, and 50 µg/ml Gentamicin) supplemented with 2% matrigel + 2 µM lapatinib (MedChemExpress, HY-50898) and refed on Day 4. On Day 6, cultures were refed with growth medium + 2% matrigel and refed this way every 4 days thereafter.

Four 15-cm plates of B2B1 cells stably expressing inducible BirA*-CSE1L or BirA*-NUP37 were prepared as overlay cultures, induced with 1 µg/ml doxycycline (Sigma, D9891) on Day 5, heterodimerized with 0.5 µM AP21967 (Takara, 635057) on Day 6, and labeled with 50 µM biotin (Sigma, B4639) on Day 7 for 24 h before lysis in RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA) plus protease and phosphatase inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM PMSF, 200 µM Na₃VO₄). Clarified lysate (3 ml) was mixed with 8 ml IAP buffer (50 mM MOPS [pH 7.2], 10 mM sodium phosphate [pH 7.4], 50 mM NaCl) and immunoprecipitated with 500 µl anti-biotin agarose (ImmuneChem, ICP0615) overnight at 4 °C. Immunoprecipitates were washed four times with PBS plus protease and phosphatase inhibitors and eluted with 0.15% trifluoroacetic acid followed by re-equilibration to pH 7 with 1 M NaOH. The eluate was re-purified with 50 µl streptavidin magnetic beads (Thermo Fisher, PI88816) for 2 h at room temperature, washed three times with TBS + 0.1% Tween 20 plus protease and phosphatase inhibitors, and boiled in Laemmli sample buffer. Samples were electrophoresed on a 10% polyacrylamide gel and fragments excised to avoid nonspecific bands at 57, 41, 27, and 16 kDa (Fig. 5b) before mass spectrometry.

Cells were washed three times with PBS(-) and subsequently cultured in the RPMI medium without IL-3 nor antibiotics for 5 h before ligand stimulation. The cells (1.0 × 10⁶ cells) were harvested by a centrifuge and the cell pellet was resuspended with 500 μl of RPMI medium with or without 50 nM AP21967 (Takara Bio) and incubated at 37℃ for 15 min. The stimulated cells (10⁶ cells) were washed twice with 2 mM Na₃VO₄ in ice-cold PBS(-). The cell pellet was lysed with 100 μl lysis buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM Na₃VO₄) and incubated on ice for 10 min. After centrifugation at 21,500 g for 10 min at 4℃, the supernatant was collected, mixed with 33 μl of 4 × Laemmli’s buffer, and boiled at 98℃ for 5 min.

DNA plasmid vectors were transfected at 60%–90% cells' confluence with 2 mg/ml, pH 7.3, polyethylenimine HCl max solution (PEI max; Polysciences). Each cell culture dish, containing 3 × 10⁶ cells, received a DNA‐PEI max mixture consisting of 10 μg total DNA along with 20 μg PEI max in Opti‐MEM Reduced Serum Media (Thermo Fisher Scientific). After 48 h transfection, HEK293T cells were treated with 1–4 μM rapamycin (LC Laboratories), 5 μM AP21967 (Takara Bio) or 99% ethanol as a vehicle for 6–24 h before lysis or fixation.

RAW 264.7 macrophages in CellView 4‐compartment glass‐bottom dishes (Greiner Bio‐One) were transfected with 0.3 μg HsLAMP1‐mCherry‐FRB* together with 0.8 μg (i) FKBP12‐(Calbindin‐2)₂‐mTagBFP2‐HA or (ii) FKBP12‐(Calbindin‐2‐EFmut)₂‐mTagBFP2‐HA or (iii) FKBP12‐(mTagBFP2)₃‐HA, per compartment using jetPRIME and allowed to express for 24 h. Cells were treated with the rapalog 250 nM AP21967 (Takara) to cause dimerization of FRB* and FKBP12 proteins, or 0.05% v/v ethanol (control) for 45 min at 37°C in DMEM. Cells were washed once with Ca²⁺‐free ECM supplemented with 1 mM EGTA, followed by two washes in Ca²⁺‐free ECM with 100 μM EGTA. Phagocytosis of Alexa 647‐conjugated 3 μm‐IgG‐beads was conducted in Ca²⁺‐free ECM supplemented with 100 μM EGTA and 250 nM AP21967 for 10 min at 37°C.