Fig 1: F-actin stabilization enhances CD36 plasma membrane localization and lipid uptake(A) Cell lysates of the CD cells indicated were analyzed by western blot for total levels of ILK and CD36.(B and C) ILK and CD36 bands were quantified by densitometry and normalized to β-actin bands. Values are expressed as a ratio of ILK/β-actin and CD36/β-actin. Values are mean ± SD with individual symbols representing a single sample. Statistical analysis: two-tail unpaired t test. (p ≤ 0.05 indicates statistical significance).(D) Plasma membrane-rich fractions (MRFs) of WT and ILK-KO CD cells were analyzed by western blot for CD36 levels and localization. Plasma membrane-associated CD36 is significantly reduced in ILK-KO cells compared to WT. Na/K ATPase and α-tubulin were used to evaluate purity of the fractions. A cytosol-rich fraction (CRF) from a control liver showing no detectable levels of Na/K ATPase, but the presence of α-tubulin, served as control.(E) CD36 bands in MRFs were quantified by densitometric analysis and expressed as CD36/Na/K ATPase ratio. Data represent mean ± SD, with individual symbols representing a single treatment. Statistical analysis: one-way ANOVA followed by multiple comparison tests. (p ≤ 0.05 indicates statistical significance).(F) Plasma MRFs of ILK-KO CD cells treated with vehicle or jasplakinolide (Jasp, 100 nM, 1 h) were analyzed by western blot for CD36 levels and localization. CD36 bands in MRFs were quantified and expressed as CD36/Na/K ATPase ratio (see E).(G) Representative images of WT (used as positive control) and ILK-KO CD cells treated with vehicle or jasplakinolide (100 nM, 1 h), stained with anti-ILK and anti-CD36 antibodies, phalloidin, and DAPI. Cell junction colocalization of CD36 and cortical F-actin is evident in jasplakinolide-treated ILK-KO CD cells compared to vehicle-treated ILK-KO cells. Scale bars, 5 μm.(H–J) Intensity plot profiles for ILK, CD36, and F-actin showing extended ILK/CD36, ILK/F-actin, and CD36/F-actin contacts in WT and jasplakinolide-treated ILK-KO CD cells, compared to vehicle-treated ILK-KO cells. Values are the mean ± SD (dotted lines) of 5 independent experiments with 4 images/cell analyzed.(K) BODIPY lipid uptake assay was performed on adherent WT and ILK-KO CD cells treated as indicated, and values are expressed as relative fluorescence unit (RFU) per μg of total cell lysates. Data represent mean ± SD, with individual symbols indicating single treatment. Statistical analysis: two-way ANOVA followed by multiple comparison tests. (p ≤ 0.05 indicates statistical significance).
Fig 2: Decreased plasma membrane-associated CD36 in hepILK-KO HFD-fed mice(A–C) Total liver lysates from control (Cnt) and hepILK-KO mice fed as indicated were analyzed by western blot for total levels of CD36 and FATP2. CD36 was not detected in SCD-fed mice and was equally upregulated in both genotypes following HFD. In contrast, levels of FATP2 were detected in both SCD-fed genotypes and remained unchanged following HFD. β-Actin and Ponceau were used to verify equal loading.(D–F) Western blots of plasma membrane-rich fractions (MRFs) showed detectable levels of CD36 only in control HFD-fed mice. In contrast, the plasma membrane-associated FATP2 levels were similar between the two genotypes and did not change in response to diet. Na/K ATPase, α-tubulin, and Ponceau were used to evaluate purity of the fractions and equal loading. A cytosol-rich fraction (CRF) from a control liver showing no detectable levels of Na/K ATPase, but the presence of α-tubulin, served as control. Densitometric quantification of the bands was performed as described in the STAR Methods. Values are expressed as a ratio of CD36 or FATP2/β-actin; or a ratio of CD36 or FATP2/Na/K ATPase. Values are mean ± SD, with symbols representing individual livers.(G) Representative images of livers from control and hepILK-KO HFD-fed mice co-stained with anti-ILK and anti-CD36 antibodies and DAPI. Cell junction colocalization of ILK and CD36 is evident in livers of HFD-fed control mice compared to hepILK-KO mice (white box). Scale bars, 10 μm.(H) Representative images of livers from control and hepILK-KO HFD-fed mice co-stained with anti-CD36 (black) and anti-Na/K ATPase (purple) antibodies and DAPI. Plasma membrane-associated CD36 (black staining) is evident in livers of HFD-fed control mice compared to hepILK-KO mice. Scale bars, 10 μm.Statistical analysis in (B), (C), (E), and (F): two-way ANOVA followed by multiple comparison tests. (p ≤ 0.05 indicates statistical significance).
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