Fig. 4. GIPR is localized to TR-containing endosomes in basal conditions and to TGN46-containing membranes upon GIP stimulation.

(A) Immunoblot of TGN-46 immunoadsorption from basal and GIP stimulated adipocytes. TGN-46 membranes were immunoisolated using anti-TGN-46 antibody in the absence of detergent. The Input (In), unbound flow-through (FL) and elution (Bd) were run on SDS-PAGE and blotted for TGN markers Syntaxin 6 and mannose-6-phosphate receptor (M-6-P cation independent) and cis-golgi marker GM-130 and for GIPR.

(B) Quantification of (A). Relative enrichment of proteins in elution were calculated by normalizing to the flowthrough.

(C) Immunoblot of GIPR immunoadsorption from basal and GIP stimulated WT adipocytes. GIPR-GFP membranes were pulled down in absence of detergent. The elution (bound) was run on SDS-PAGE together with input and unbound (FL) and blotted for GIPR, TR and TGN-46.

(D) Quantification of (C). Relative amounts of TR or TGN in the elution were calculated by normalizing to input and GIPR in the IP.

(E) Quantification of GIP induced GIPR downregulation in syntaxin-6 (Sx-6) knockdown adipocytes. Two different siRNAs were used. Both knockdowns show a decrease in GIPR downregulation showing that it depends on Sx-6. Data are averages ± SD of 3 independent experiments., P<0.05.

(F) Exocytosis rate constants for TR and TGN vesicles compared with GIPR in basal and GIP stimulated condition. GIPR k_e values are plotted from data in Fig. 1.

(G) Schematic representation of GIPR recycling from the ERC (TR-containing endosomes) or TGN compartments with associated rate constants. k_i, internalization rate constant, ‘k_ERC’, rate from the ERC; ‘k_TGN’, rate from TGN; and ‘k_sort’, GIPR sorting rate from ERC to the TGN. Additional connectivities between these compartments did not significantly improve the performance of this model.

(H) Calculation of GIPR sorting rate constant (K_sort) by using equation 1 (supplementary methods) and the model in Fig. 4G. Data from Fig. 1F was used. Inset shows the value of ‘r’ calculated under basal or upon GIP stimulation.

(I) GIPR as a fraction of total in different compartments under basal or GIP stimulated conditions. The values were calculated from (F).

(J) And (K) Immunoblot of GIPR immunoadsorption from basal and GIP stimulated βA2 KD adipocytes.