α rat alexafluor 647

HEK293T were transfected with specified plasmids. 17–24 hr post transfection, HEK293T cells were washed with 1 mM CaCl₂/PBS, lifted off the dish with 2 mM EDTA/PBS, pelleted and lysed by incubating in lysis buffer (150 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.5% NP-40, 1x PhosSTOP phosphatase inhibitor (Roche), 1x HALT protease inhibitor (Thermo Fisher Scientific) for 30 min, and bath sonicated in ice for 3 min. Cell debris was pelleted at 18,000 rcf for 10 min. Cell lysate were precleared with 15 μl of GFP-Trap (Chromotek) for 30 min at 4°C, and incubated with 15 μl of fresh GFP-Trap beads overnight at 4°C. The beads were washed with lysis buffer five times, before boiled in Laemmli sample buffer and separated on 4–20% acrylamide gradient gels by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and probed with primary antibodies, α-Grb2 (1:5000, Clone 81/Grb2, BD Biosciences), α-tubulin (1:5000, Clone YL1/2, Thermo), α-pTyr (1:2000, Phospho-Tyrosine (P-Tyr-1000) MultiMab Rabbit mAb mix #8954, Cell Signaling Technology), α-GFP (1:10000, Clone 3E6, Life Technologies or 1:5000, A21312, Life Technologies), and secondary antibodies, α-mouse HRP (1:10,000, Upstate Biotechnology or 1:5000, Jackson Labs), α-rabbit HRP (1:5000, 65–6120, Thermo Fisher), α-rat AlexaFluor 647 (1:5000, Life Technologies). Western blots were imaged on ChemiDoc (Bio-Rad).

HEK293T were transfected with p14 WT, p14 Y116F/N118A, and p14 Δcyto. 18 hr post transfection, the cells were washed with 1 mM CaCl₂/PBS, lifted off the dish with 2 mM EDTA/PBS. Cells were pelleted at 200 rcf for 5 min and re-suspended in fractionation buffer (20 mM HEPES, 10 mM KCl, MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM TCEP, 1x HALT protease inhibitor(Thermo Fisher Scientific)). The cell suspension lysed with five freeze/thaw cycles. Nuclei were pelleted via centrifugation (700 rcf, 5 min), and mitochondria were pelleted at 10,000 rcf, 5 min. The supernatant was then centrifuged at 100,000 rcf for an hour at 4°C to separate the membrane and cytoplasmic fraction. The membrane pellet was washed once in fractionation buffer and re-centrifuged at 100,000 rcf for an hour. The cell lysate, cytoplasmic fraction, and membrane pellet was boiled in Laemmli sample buffer, and separated on 4–20% acrylamide gradient gels by SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and probed with primary antibodies, α-tubulin (1:5000, Clone YL1/2, Thermo Fisher Scientific), α-GFP (1:5000, A-21312, Life Technologies), and secondary antibodies, α-rabbit HRP (1:5000, 65–6120, Thermo Fisher) and α-rat AlexaFluor 647 (1:5000, Life Technologies). Western blots were imaged on a ChemiDoc (Bio-Rad).