Extended Data Fig. 1. RalA protein content and activity are increased in obese adipocytes.

a, Immunoblotting for RalA in mature adipocytes isolated from iWAT (n = 3) or eWAT (n = 4) of age-matched CD-fed (lean) mice and HFD-fed (obese) mice. b, Representative immunoblotting for active RalA (aRalA) in iWAT (upper panel) or eWAT (lower panel) of age-matched CD-fed mice (n = 3) and HFD-fed mice (n = 2). c, RalA mRNA expression and quantified protein levels in BAT of age-matched CD-fed and HFD-fed mice (n = 8). d, Immunoblotting of RalA in inguinal mature adipocyte fraction, eWAT, BAT, and liver from lean mice (n = 3). e, In vivo and in vitro activation of RalA by insulin in adipose tissue of Rala^f/f (saline n = 3, insulin n = 2) and Rala^AKO (n = 4) mice and primary adipocytes. 0.5U/kg or 100 nM insulin was injected or treated for 5 min or indicated time. f, Basal in vivo glucose uptake in 6 hrs fasted CD-fed mice injected with 10μCi [¹⁴C]-2deoxyglucose for 30 min (Rala^f/f n = 7, Rala^AKO n = 5). g, Plasma insulin levels before and 30 min after glucose injection (Rala^f/f n = 9, Rala^AKO n = 5). h, Insulin stimulated in vivo glucose uptake in CD-fed mice injected with 1.2 g/kg glucose and 10 μCi [³H]-2-deoxy-glucose for 30 min (Rala^f/f n = 6, Rala^AKO n = 9). i, Immunoblotting of RalA in BAT (upper panel) and eWAT (lower panel) of Rala^f/f and Rala^BKO mice (n = 3). j, Basal in vivo glucose uptake in 6 hrs fasted CD-fed mice injected with 10 μCi [¹⁴C]-2-deoxy-glucose for 30 min (Rala^f/f n = 7, Rala^BKO n = 5). k, Plasma insulin levels before and 30 min after glucose injection (Rala^f/f n = 5, Rala^BKO n = 7). l, Insulin stimulated in vivo glucose uptake in CD-fed mice injected with 1.2 g/kg glucose and 10 μCi [³H]-2-deoxy-glucose for 30 min (Rala^f/f n = 5, Rala^BKO n = 7). m, Representative immunostaining of endogenous RalA and GLUT4 in primary adipocytes treated with insulin (100 nM) or vehicle for 30 min, scale bar = 15 μm. (n = 3 biological samples). n, Representative immunoblotting of RalA, GLUT4, IRAP and Na + /K + -ATPase proteins in plasma membrane fraction of primary adipocytes treated with vehicle or insulin (100 nM) for 30 min. (n = 3 biological samples). o, 2-deoxy-glucose (2-DG) uptake in primary adipocytes treated with insulin (100 nM) or vehicle for 30 min (n = 3 biological samples). p, Immunoblotting of phosphor-Akt (S473), total Akt and GAPDH in primary adipocytes treated with or without insulin (100 nM) for 15 min. (n = 3 biological samples). The data are shown as the mean ± SEM, *P < 0.05, **P < 0.01 by two-tailed Student’s T-test (h, l, o).

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