Nb400 144

Tissue lysates were separated by SDS-PAGE and transferred to 0.45 μm polyvinylidene difluoride membranes (Immobilon, Millipore. Merck KGaA, Darmstadt, Germany). Blots were incubated with antibodies directed against CD36 (NB400-144), ATF6 (NBP1-40256), and CHOP (NB600-1335) purchased from Novus Biologicals (Bio-Techne LD-R&D Systems Europe Ltd., Abingdon, UK). Equal loading of protein in each lane was verified by β-actin (A5441, Sigma–Aldrich, Merck KGaA, Darmstadt, Germany).

Cell-surface delivery of CD36 was quantified as previously described. [22 (link)] Briefly, L6-GLUT4myc myoblasts were plated at a cell density of 3 × 10⁵ cells/well into 8-well chamber slides and allowed to attach overnight. Cells were then treated with insulin or ionomycin as described above in the presence of the exocyst inhibitor endosidin 2 (ES2) or DMSO as control. Heterozygous EXOC5 knockout (ΔEXOC5) cells were stimulated with 100 nM insulin or 1 μM ionomycin in parallel with the appropriate controls, as described above. Following fixation with 4% paraformaldehyde and blocking with 5% BSA in PBS, surface CD36 was detected using a primary antibody targeting the extracellular domain of CD36 (Rabbit, Cat# NB400-144, Novus) diluted 1:100 in 5% BSA in PBS. Following subsequent permeabilization in 0.1% Triton X-100 for 10 min, the cells were incubated with an anti-β-tubulin primary antibody (Mouse, Proteintech, sc-5274) diluted 1:100 as control. Cells were then incubated with IRDye secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) diluted at 1:15,000. The chamber slides were scanned using the Odyssey Clx scanner. Densitometry was then performed with Image Studio software (LI-COR Biosciences). Surface CD36 signal intensity was normalized with total β-tubulin levels measured.

Paraffin‐embedded liver biopsy sections (4 µm thick) were immunostained with a primary rabbit antibody against HIF2α (ab199, Abcam) ²⁸ or CD36 (NB400‐144, Novus) diluted to 1:50 and 1:200 respectively, using the DAKO EnVision™+ System (DAKO, Glostrup, Denmark) as described by the manufacturer. Liver tissue area occupied by nuclear HIF2α or CD36‐positive cells was quantified from the images taken using an optical microscope Nikon Eclipse E400 (Nikon). Image analysis procedures were performed with the FIJI software (NIH). Values were obtained in six different lobular areas where hepatocytes are the predominant cell type. The average value was considered as nuclear HIF2α or CD36 expression index for each liver biopsy sample, and expressed as percentage of positive nuclei or arbitrary units respectively, which reflects the intensity of the immunostaining.

Immunohistochemical (IHC) staining was performed according to a previously reported standard protocol.^{18 (link)} CD36 antibody (NB400-144, rabbit, diluted 1:300; Novus Biologicals) and CAIX antibody (NB100-417, rabbit, diluted 1:500; Novus Biologicals) were used for the IHC assessment. Based on intensity, the staining was graded/scored as no staining (0), weak staining (1), moderate staining (2), and strong staining (3). The number of positively stained GC cells was divided into the following four ranges (proportion score): ≤5% (0), 6–25% (1), 26–50% (2), and >51% (3). The final staining score was calculated using the following formula: overall score = intensity score × proportion score. A final score of ≤4 was defined as negative staining and >4 as positive staining.

Paraffin‐embedded liver biopsy sections (4 μm thick) were co‐incubated with an anti‐CD36 antibody (NB400‐144, Novus) and anti‐N‐cadherin antibody (BP1‐48309, Novus), diluted to 1:200 and 1:25 respectively, following with the appropriated conjugated secondary antibodies Alexa Fluor® 568 goat anti‐rabbit IgG (A11011, Life Technologies) or Alexa Fluor® 488 goat anti‐mouse IgG (A11029, Life Technologies). The immunofluorescence‐mounting medium used was Fluoromont G® (BioNova cientifica). Representative images were taken using a confocal microscope Leica TCS SP5 X (Leica, Barcelona, Spain).

Tissues were homogenized in ice-cold sigma buffer (Sigma Aldrich #C3228) with protease and phosphatase inhibitor cocktails. Enterocytes were harvested from the proximal segment of the small intestine (proximal 10 cm) after the intestine was rinsed with cold PBS to remove luminal contents. Homogenates were centrifuged at 1000 × g for 15 minutes at 4 °C and supernatants were collected. Protein concentration was determined by DC protein assay kit (Bio-Rad #5000116) and 40 μg of protein were resolved by electrophoresis on SDS-8% polyacrylamide gels. Proteins were transferred to PVDF membranes and were detected by the following primary antibodies: TIMP4 (Abcam #ab51207) and CD36 (Novus Biologicals #NB400-144). Respective HRP-conjugated secondary antibodies were used to detect immunoreactivity. Immunoreactive proteins were visualized using the ECL kit and band intensities were quantified by ImageJ software. Coomassie blue-stained gel was used as internal loading controls.

The expression of CD36 in paraffin-embedded tumoral and peritumoral tissues was analyzed by immunohistochemistry using the CD36 antibody NB400-144 (Novus Biologicals, Littleton, CO, USA). Each sample, with a 5-µm thick section, was processed using the BenchMark Ultra Ventana. The deparaffinization of the samples (72 °C for 16 min) was followed by antigen retrieval for 56 min at pH 8 at 100 °C (Ultra CC1). Endogenous peroxidase activity was then blocked for 5 min. The incubation with CD36 primary antibody (36°C for 36 min) was followed by incubation with OptiView HRP Multimer (36 °C, 8 min) and OptiViewDAB (36 °C, 8 min). Counterstaining was carried out with hematoxylin (36 °C, 16 min) and post-counterstaining with bluing reagent (36 °C, 8 min). The slides were examined by a pathologist using a DM750 light microscope (Leica Microsystems GmbH). CD36 expression levels were evaluated by calculating the percentage of positive tumoral cells. The images were acquired using the Axiocam 305 Color (ZEISS).