primary antibodies targeting cd36  (Thermo Fisher)

Journal: Cells

Article Title: Imeglimin Inhibits Macrophage Foam Cell Formation and Atherosclerosis in Streptozotocin-Induced Diabetic ApoE-Deficient Mice

doi: 10.3390/cells14070472

Figure Lengend Snippet: Effects of imeglimin on the expression of ABCA1, ABCG1, and CD36 proteins in ox-LDL/HG-treated THP1 macrophages. THP-1 macrophages were pretreated with imeglimin and then exposed to 100 μg/mL ox-LDL and 30 mM glucose. ( A ) Western blot analysis showing the expression of ABCA1, ABCG1, and CD36 proteins; ( B ) Relative protein expression levels of ABCA1, ABCG1, and CD36 (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01. HG, high glucose; IME, imeglimin; oxLDL, oxidized lowdensity lipoproteins.

Article Snippet: Membranes were blocked using 5% bovine serum albumin at room temperature for 1 h, followed by overnight incubation at 4 °C with primary antibodies targeting CD36 (PA1-16813, 1:1000, Thermo Fisher Scientific), ABCA1 (NB400-105, 1:1000, Novus Biologicals, Littleton, CO, USA), ABCG1 (NB400-132, 1:1000, Novus Biologicals), AMPK (2532, 1:1000, Cell Signaling Technology), phosphorylated AMPK (pAMPK, 2535, 1:1000, Cell Signaling Technology), and β-actin (ab8227, 1:10,000, Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: Imeglimin Inhibits Macrophage Foam Cell Formation and Atherosclerosis in Streptozotocin-Induced Diabetic ApoE-Deficient Mice

doi: 10.3390/cells14070472

Figure Lengend Snippet: Effect of imeglimin on the expression of ABCA1, ABCG1, and CD36 in the presence of the AMPK inhibitor compound C. THP-1-derived macrophages were pretreated with compound C, incubated with 100 μM imeglimin for 24 h, and exposed to 100 μg/mL oxLDL and 30 mM glucose. ( A ) Western blot analysis showing the expression of total AMPK (tAMPK) and phosphorylated AMPK ( p -AMPK) proteins; ( B ) Relative protein expression levels of t-AMPK and pAMPK (quantified using ImageJ); ( C ) Western blot analysis showing the expression of ABCA1, ABCG1, and CD36 proteins; ( D ) Relative protein expression levels of ABCA1, ABCG1, and CD36 (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001. HG, high glucose; IME, imeglimin; ox-LDL, oxidized low-density lipoproteins.

Article Snippet: Membranes were blocked using 5% bovine serum albumin at room temperature for 1 h, followed by overnight incubation at 4 °C with primary antibodies targeting CD36 (PA1-16813, 1:1000, Thermo Fisher Scientific), ABCA1 (NB400-105, 1:1000, Novus Biologicals, Littleton, CO, USA), ABCG1 (NB400-132, 1:1000, Novus Biologicals), AMPK (2532, 1:1000, Cell Signaling Technology), phosphorylated AMPK (pAMPK, 2535, 1:1000, Cell Signaling Technology), and β-actin (ab8227, 1:10,000, Abcam, Cambridge, UK).

Techniques: Expressing, Derivative Assay, Incubation, Western Blot

Journal: Cells

Article Title: Imeglimin Inhibits Macrophage Foam Cell Formation and Atherosclerosis in Streptozotocin-Induced Diabetic ApoE-Deficient Mice

doi: 10.3390/cells14070472

Figure Lengend Snippet: Effects of imeglimin on AMPK activity and ABCA1, ABCG1, and CD36 protein expression in diabetic ApoE -/- mice. ( A ) Western blot analysis showing the expression of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the liver; ( B ) Relative protein expression levels of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the liver (quantified using ImageJ); ( C ) Western blot analysis showing the expression of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the aorta; ( D ) Relative protein expression levels of pAMPK, tAMPK, ABCA1, ABCG1, and CD36 in the aorta (quantified using ImageJ). Results are expressed as the means ± SEMs of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were blocked using 5% bovine serum albumin at room temperature for 1 h, followed by overnight incubation at 4 °C with primary antibodies targeting CD36 (PA1-16813, 1:1000, Thermo Fisher Scientific), ABCA1 (NB400-105, 1:1000, Novus Biologicals, Littleton, CO, USA), ABCG1 (NB400-132, 1:1000, Novus Biologicals), AMPK (2532, 1:1000, Cell Signaling Technology), phosphorylated AMPK (pAMPK, 2535, 1:1000, Cell Signaling Technology), and β-actin (ab8227, 1:10,000, Abcam, Cambridge, UK).

Techniques: Activity Assay, Expressing, Western Blot

Journal: Cells

Article Title: The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells

doi: 10.3390/cells11152440

Figure Lengend Snippet: The exocyst is recruited to CD36 vesicles in response to stimuli. ( A , C ) Confocal images of control and insulin− or ionomycin−induced L6−GLUT4myc rat skeletal myoblasts immunostained for exocyst subunit Exoc5 and CD36 show an increased colocalization of CD36 and Exoc5 in skeletal myoblasts upon treatment. ( B , D ) Colocalization is expressed as average coefficients of correlation (Pearson’s). ( E ) Microscopic images and quantification of a PLA assessing EXOC5 and EXOC7 subunit proximity in L6 GLUT4myc myoblasts show an increase in exocyst subunit proximity following ionomycin treatment. In the presence of exocyst inhibitor ES2, ionomycin fails to stimulate exocyst assembly. ( F , G ) Microscopic images and quantification of a PLA evaluating EXOC5 subunit proximity with CD36 in L6 GLUT4myc myoblasts following insulin or ionomycin treatment in the presence or absence of ES2. Both treatments stimulate EXOC5 recruitment to CD36, as demonstrated by an increase in PLA signals—shown in red or green. The DAPI stain is blue. Images are representative of at least three independent experiments. Data are presented as mean ± S.D normalized to controls. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0005; ns: p > 0.05. Scale bars: 5 µm ( A , C ); 10 µm ( E – G ).

Article Snippet: 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.

Techniques: Control, Staining

Journal: Cells

Article Title: The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells

doi: 10.3390/cells11152440

Figure Lengend Snippet: Disrupted exocyst function impairs CD36 membrane delivery. Representative in−cell Western analysis shows that insulin stimulates higher cell−surface levels of CD36 in L6 GLUT4myc myoblasts ( A ) and differentiated myotubes ( B ), an effect significantly reduced in the presence of the exocyst inhibitor ES2. Signal intensities of the entire surface of the chamber slide wells were measured with a fluorescent scanner. Surface CD36 levels were normalized to β−tubulin levels measured following permeabilization and immunostaining. Representative in−cell Western analysis and quantitation reveal elevated cell surface CD36 levels in L6 GLUT4myc myoblasts ( C ) and differentiated myotubes ( D ) treated with ionomycin. This response to ionomycin treatment is blunted in the presence of ES2. Quantification of relative surface CD36 levels in wild−type and ΔEXOC5 myoblasts treated with insulin ( E ) or ionomycin ( F ) indicates that exocyst disruption impairs membrane trafficking of the FFA translocase. Data are means ±S.D. in percent ratios relative to the untreated or wild−type controls. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0005; ns: p > 0.05. The images and data presented are representative of three independent measurements.

Article Snippet: 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.

Techniques: Membrane, In-Cell ELISA, Immunostaining, Quantitation Assay, Disruption

Journal: Cells

Article Title: The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells

doi: 10.3390/cells11152440

Figure Lengend Snippet: Identifying Rab GTPases associated with the exocyst and CD36 in response to insulin. ( A – C ) Microscopic images and quantification of a PLA evaluating proximity of Rab8a, Rab11 and Rab14 with CD36 in L6 GLUT4myc myoblasts show higher association rates of all three Rabs with CD36 following insulin treatment. In the presence of ES2, insulin fails to trigger such an increase in target protein proximity. ( D – F ) Microscopic images and quantification of a PLA evaluating proximity of Rab8a, Rab11 and Rab14 with EXOC5 in L6 GLUT4myc myoblasts following insulin treatment demonstrate Exoc5 recruitment to Rabs 8a, 11 and 14. This recruitment is inhibited by treatment with ES2. PLA signals are shown in red; DAPI stain is in blue. Images are representative of three independent experiments. Scale bars: 10 μm. Data are presented as means ± S.D normalized to controls. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0005; ns: p > 0.05.

Article Snippet: 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.

Techniques: Staining

Journal: Cells

Article Title: The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells

doi: 10.3390/cells11152440

Figure Lengend Snippet: Identifying Rab GTPases associated with the exocyst and CD36 in response to contraction-mimetic treatment. ( A – C ) Microscopic images and quantifications of PLAs demonstrate increased proximity of Rab8a, Rab11 and Rab14 with CD36 in L6 GLUT4myc myoblasts following ionomycin treatment. This effect of ionomycin was diminished in the presence of the ES2 inhibitor. ( D – F ) Microscopic images and quantifications of PLAs evaluating proximity of EXOC5 with Rab8a, Rab11 and Rab14 in L6 GLUT4myc myoblasts in response to ionomycin treatment reveal elevated signal-to-nuclei ratios, indicating a rise in physical proximity of the target proteins. Exocyst inhibition by ES2 blocks this ionomycin-stimulated association of Exoc5 with Rabs 8a, 11 and 14. PLA signals are shown in red or green; DAPI stain is in blue. Images are representative of three independent experiments. Scale bars: 10 µm. Data are presented as means ± S.D normalized to controls. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0005; ns: p > 0.05.

Article Snippet: 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.

Techniques: Inhibition, Staining

Journal: Journal of Advanced Research

Article Title: Soluble Trem2 is a negative regulator of erythrophagocytosis after intracerebral hemorrhage in a CD36 receptor recycling manner

doi: 10.1016/j.jare.2022.03.011

Figure Lengend Snippet: CD36 receptor recycling is a target of sTrem2 during erythrophagocytosis. (A) Representative histogram displaying the distribution of CD36 and SIRPα in microglia/macrophage treated with AAV-Ctrl or AAV-sTrem2 3 days post ICH (top). Quantification of the mean fluorescence to indicate the cell surface marker change (bottom). n = 3 per group, **p < 0.01 versus AAV-Ctrl by Student’s t test. (B, C) Using an established receptor recycling assay, CD36 receptor recycling was analyzed in magnetic beads isolated cells from AAV-Ctrl and AAV-sTrem2 mice 3 days post ICH. Scale bar = 10 μm. The positive pixels in cells were quantified as recycling CD36. N = 4 per group, ***p < 0.001 by Mann–Whitney U test. (D) CD11b + cells isolated from AAV-Ctrl or AAV-sTrem2 brains 3 days after ICH were used for surface expression (left), internalization (middle) and recycling (right) of CD36 assay by flow cytometry. N = 5 per group, **p < 0.01, ***p < 0.001 versus AAV-Ctrl by Student’s t test. (E) Analysis of CD36 surface expression (left), internalization (middle) and recycling (right) in CD11b + from 1 day post-ICH brains by flow cytometry. n = 3 per group **p < 0.01 versus AAV-Ctrl by Student’s t test. (F) CD36 receptor recycling was analyzed in primary microglia and BV2 under sTrem2 and vehicle treatment (Hb) after hemoglobin stimulation. Scale bar = 50 μm. n = 3 per group, *p < 0.05, **p < 0.01 versus Hb by Student’s t test.

Article Snippet: Cells were blocked with 10% donkey serum in DMEM for 15 min, and primary antibodies targeting CD36 (1:100, ab23680, Abcam) were incubated with cells for 1 h at a dilution of 1:100.

Techniques: Fluorescence, Marker, Magnetic Beads, Isolation, MANN-WHITNEY, Expressing, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: Soluble Trem2 is a negative regulator of erythrophagocytosis after intracerebral hemorrhage in a CD36 receptor recycling manner

doi: 10.1016/j.jare.2022.03.011

Figure Lengend Snippet: Vps35 engages in sTrem2-mediated microglial CD36 receptor recycling after ICH. (A) CD36 and receptor recycling associated protein: Beclin1, Vps35 from the CD11b + cells were analyzed and quantified by Western blotting 3 days after ICH in AAV-Ctrl or AAV-sTrem2 mice. n = 3 per group, *p < 0.05, **p < 0.01 versus AAV-Ctrl by Student’s t test. (B) CD36, Beclin1, and Vps35 from CD11b + cells from 1 day post-ICH brain tissues. n = 3 per group, *p < 0.05 versus AAV-Ctrl by Student’s t test. (C) Primary microglia were treated with sTrem2 protein and hemoglobin stimulation. CD36, Beclin1, and Vps35 levels were determined 24 h after treatment. n = 3 per group, **p < 0.01 versus Vehicle by Student’s t test. (D) Representative images of CD36 receptor recycling in BV2 cells transfected with Vps35 or control lentivirus with/without sTrem2 protein administration under hemoglobin stimulation. Scale bar = 50 μm. (E, F) Quantification of recycled receptors in BV2 microglia of each group. n = 3 per group, *p < 0.05 versus Vehicle by Mann–Whitney U test. (G) Histogram representing the distribution of RBC-positive BV2 microglia treated with control or Vps35 and vehicle or sTrem2 protein (left). The phagocytic index was analyzed to indicate erythrophagocytosis. n = 3 per group, *p < 0.05 by Student’s t test.

Article Snippet: Cells were blocked with 10% donkey serum in DMEM for 15 min, and primary antibodies targeting CD36 (1:100, ab23680, Abcam) were incubated with cells for 1 h at a dilution of 1:100.

Techniques: Western Blot, Transfection, Control, MANN-WHITNEY

Journal: Journal of Advanced Research

Article Title: Soluble Trem2 is a negative regulator of erythrophagocytosis after intracerebral hemorrhage in a CD36 receptor recycling manner

doi: 10.1016/j.jare.2022.03.011

Figure Lengend Snippet: sTrem2 facilitates lysosomal degradation of unrecycled CD36. (A, B) Representative images and quantification showing colocalization of recycling CD36 (red) and Lamp1 (green) in primary cultured microglia (gray) under sTrem2 administration. Scale bar = 10 μm. n = 3 per group, *p < 0.05 versus Vehicle by Student’s t test. (C, D) Representative images and quantification of CD36 accumulation in BV2 microglial lysosome after control or Vps35 lentivirus infection with/without sTrem2 protein treatment. Scale bar = 10 μm. n = 3 per group. (E, F) BV2 cells were transfected with control or Vps35, and subsequently purified by density gradient centrifugation to obtain lysosome fraction. CD36 accumulated in lysosome of control or Vps35 treated BV2 with/without sTrem2 was analyzed by Western blotting. n = 3 per group, **p < 0.01, ***p < 0.001 versus Vector + Vehicle by one-way ANOVA and Tukey’s post hoc test. (G, H) Representative images showing CD36 accumulation in CD11b + cells lysosome from AAV-Ctrl and AAV-sTrem2 infected mice 3 days post ICH. Scale bar = 10 μm, n = 3 per group, *p < 0.05 versus AAV-Ctrl by Student’s t test.

Article Snippet: Cells were blocked with 10% donkey serum in DMEM for 15 min, and primary antibodies targeting CD36 (1:100, ab23680, Abcam) were incubated with cells for 1 h at a dilution of 1:100.

Techniques: Cell Culture, Control, Infection, Transfection, Purification, Gradient Centrifugation, Western Blot, Plasmid Preparation