Alexa fluor plus 680

S2 cells were transfected with pMT-N-EGFP, pMT-Flag-Ubi, and pMT-Dx-V5 (WT or ΔRF). After 48 h, 1 mM CuSO₄ was added to induce protein expression, followed by incubation at 18°C for 24 h. The cells were lysed by lysis buffer (50 mM Tris-HCl, pH7.5, 125 mM NaCl, 5% glycerol, 0.5% NP-40, 1.5 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM N-ethyl-maleimide, 10 μM MG132, and Halt Protease and Phosphatase Inhibitor Cocktail [Thermo Fisher Scientific]), and pulled-down with GFP-Trap (ChromoTek). The immunoprecipitated samples and 10% lysates were separated on NuPage 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Merck). Primary antibodies used for Western blotting were rabbit anti-GFP (50430-2-AP, 1:5,000; Proteintech), mouse anti-Flag (M2, 1:5,000; Merck), mouse anti-V5 (OASA04489, 1:5,000; Aviva System Biology), and mouse anti-Peanut ((Cat# 4C9H4 anti-peanut, RRID:AB_528429, 1:10,000; DSHB), and staining was detected by LI-COR Odyssey imaging system with anti-mouse Alexa Fluor Plus 800 (Cat# A32730, RRID:AB_2633279; Thermo Fisher Scientific) and anti-rabbit Alexa Fluor Plus 680 (Cat# A32734, RRID:AB_2633283; Thermo Fisher Scientific) secondary antibodies, used at 1:10,000.

For immunocytochemistry, approximately 2 × 10⁵ of RKO cells were seeded into plastic coverslips and cultured overnight. FAM-RKOpep was diluted in PBS 1X (50 µM) and incubated with adherent RKO cells for 1 h at 4 °C. After rinsing with PBS, the cells were fixed with 4% of PFA for 40 min and then permeabilized with 0.01% Triton-X (Merck) for 5 min. Afterwards, non-specific binding sites were blocked with TBS-T 1X containing 5% BSA for 30 min at 4 °C, and next exposed overnight at 4 °C to the primary antibody (dilution in TBS-T with 5% BSA) MCT1 (1:200 dilution, AB3538P, Merck). After rinsing with PBS 1X, the cells were incubated with anti-rabbit (#A-11011, Thermo Scientific) secondary antibody coupled to Alexa Fluor Plus 680 (1:500 dilution in TBS-T with 5% BSA) for 90 min at room temperature. After being washed with PBS 1X, the cells were stained with Vectashield mounting media containing DAPI. The images were acquired by an Olympus BX51 microscope incorporated with a high-sensitivity camera Olympus DP72 at 100X magnification.

Lung tissue was frozen with liquid nitrogen and embedded in Optimal cutting temperature compound (4583, Sakura) and sectioned at 10 μm thickness, followed by immediate fixation in 96% ice‐cold ethanol (01396, Histolab) for 30 minutes followed by 3× 5 minute washing in PBS (14190‐144, Gibco). Tissue staining was then performed using BlockAid^™ (B10710, Invitrogen), followed by overnight incubation in 4°C with primary antibodies against HIF1a (ab82832, abcam) or HIF2a (NB100‐122, Novus) diluted 100 times. Detection was performed by staining with anti‐Rabbit IgG Alexa Fluor Plus 680 (A32802, Invitrogen) at room temperature for 1 hour followed by washing with PBS and mounting with ECTASHIELD^® Antifade Mounting Medium with DAPI (H1200, Vector Laboratories).

FPLC was performed as previously described^{40 (link)}. Serum (50 µL) from two representative noncarrier or carrier siblings was injected into an ÄKTA Go FPLC (GE Healthcare). A Superdex 200 10/300 GL column (GE Healthcare) was used to separate adiponectin complexes in HEPES/Ca²⁺ buffer (25 mM HEPES; 150 mM NaCl; and 1 mM CaCl₂, pH 7.4). 250 µL fractions were collected over a 20 mL retention volume. The retention volumes found to contain adiponectin were then used to run Western blots to determine HMW, LMW and trimeric adiponectin. Samples were run on an SDS-Page gel (BioRad Criterion TGX) after being reduced in Laemmli and 355 mM 2-mercaptoethanol and boiled for 10 min. The gel was transferred to PVDF membrane (BioRad). The membranes for each patient were blocked in 5% BSA then probed for adiponectin overnight with rabbit polyclonal anti-adiponectin at a 1:1000 dilution (Abcam, ab75989). There were washed then stained with goat-anti-rabbit AlexaFluor Plus 680 (Invitrogen) at a 1:4000 dilution. Then washed again and imaged on a ThermoFisher iBright system. Western blots derive from the same experiment and were processed in parallel.