Donkey anti goat 680rd

The 1 × 10⁶ cells were collected, washed once with PBS, and lysed using RIPA buffer containing 10× protease inhibitor cocktail (Roche). Lysates were centrifuged at 20,000g for 20 min at 4°C. Samples were reduced using 4× SDS sample loading buffer and 10× reducing buffer, and then boiled at 95°C for 3 min. Exosomes isolated by differential centrifugation were resuspended directly in sample loading buffer and boiled. Protein was run at 120 V for 1 h using a 2% to 12% gradient protein gel (Thermo Fisher Scientific). Proteins were transferred to an Immuno-Blot polyvinylidene fluoride (PVDF) membrane using Bio-Rad wet transfer apparatus for 1 h at 120 V. Membranes were blocked with LICOR blocking buffer for 1 h and incubated with mouse anti-β-actin (Sigma) at 1:1,000, polyclonal goat anti-ACE2 (R&D AF933) at 1:1,000, polyclonal rabbit anti-ACE2 at 1:1,000 (Abcam, cat# ab15348), monoclonal mouse anti-CD63 (Abcam, cat# ab217345), or monoclonal mouse anti-CD9 (R&D, cat# MAB25292) at 1:1,000 overnight at 4°C. Membranes were washed 3 times for 5 min with PBS-T and probed with donkey anti-rabbit LICOR IRdye 800CW, donkey anti-goat 680RD, goat anti-mouse 800CW, or goat anti-rabbit LICOR IRdye 800CW/680RD (LICOR Biosciences) for 1 h at RT. Membranes were imaged on a LICOR Odyssey CLX imaging system.

AICAR and 4‐Nitrobenzylthioinosine (NBMPR) were purchased from Tocris (Abingdon, UK). Dibutyryl cAMP, uridine and metformin were purchased from Sigma (Dorset, UK). LC‐MS materials were obtained from Fisher Scientific (Loughborough, UK) unless otherwise stated. Glucagon was obtained from Novo Nordisk (Bagsvaerd, Denmark). 8CPT‐cAMP was purchased from Calbiochem (Nottingham, UK) and 8CPT‐2‐Methyl‐O‐cAMP (8CPT‐2MeO‐cAMP) was purchased from Biolog Life Sciences Institute (Bremen, Germany). Donkey anti‐goat 680RD, donkey anti‐mouse 680RD, donkey anti‐rabbit 800CW secondary antibodies were purchased from LI‐COR Biosciences, UK. The GAGO glucose oxidase kit was purchased from Sigma. [5,6‐³H] uridine was purchased from Perkin Elmer (Beaconsfield, UK). pAMPK (Thr172) and total ACC antibodies were purchased from Cell Signalling Technologies (Leiden, UK). Total AMPK was purchased from Abcam (Cambridge, UK) and actin was purchased from Proteintech (Manchester, UK). pACC was made in‐house by Division of Signal Transduction Therapy (University of Dundee).