Journal: Journal of Lipid Research

Article Title: High-density lipoprotein attenuates lipopolysaccharide-induced IL-1β activation via scavenger receptor class B type 1

doi: 10.1016/j.jlr.2025.100858

Figure Lengend Snippet: SRB1 is required for LPS-stimulated IL-1β activation. A: Inhibition of SRB1 attenuates LPS-mediated IL-1β activation. RAW264.7 cell were incubated with vehicle or LPS (1 μg/ml) with or without HDL (250 μg/ml) with or without BLT-1 (1 μM) for 4h. Cell lysates were harvested and processed for western blotting for detection of pro IL-1β protein expression. The protein level of β-actin was examined as a loading control. B: Protein levels of pro IL-1β were quantitated by densitometric analysis, normalized to β-actin (mean ± SD, n = 3), ∗ P < 0.05 comparison between IL-1β alone and IL-1β+HDL; and comparison between IL-1β+HDL and IL-1β+HDL+BLT-1, ∗∗∗ P < 0.001comparison between IL-1β alone and IL-1β+HDL+BLT-1. C: Supernatants collected from B were processed for ELISA for detection of active IL-1β production using a mouse ELISA kit, ∗ P < 0.05 comparison between IL-1β+HDL and IL-1β+HDL+BLT-1, ∗∗∗ P < 0.001 comparison between IL-1β alone and IL-1β+HDL; and comparison between IL-1β alone and IL-1β+HDL+BLT-1. D: Knockdown of SRB1 attenuates LPS-mediated IL-1β activation. RAW264.7 (scramble) or RAW264.7 (shSRB1) cells were incubated with vehicle of LPS (1 μg/ml) with or without HDL (250 μg/ml) for 4 h. Cell lysates were harvested and processed for western blotting for detection of the protein expression of SRB1, pro IL-1β, and β-actin. E: Supernatants collected from C were processed for ELISA for detection of active IL-1β production using a mouse ELISA kit, ∗∗∗ P < 0.001. F: Knockdown of SRB1 inhibits LPS uptake by RAW264.7. RAW264.7 (scramble) or RAW264.7 (shSRB1) cells were incubated with fluorescently-labeled LPS (1 μg/ml) in the presence or absence of HDL (250 μg/ml) for 4 h, followed by eBioscience™ Fixable Viability Dye eFluor™ 450 staining for 30 min at room temperature. Cells that engulf fluorescently-labeled LPS were captured by flow cytometry. G: Quantitative results of flow cytometry, n = 3, ∗∗∗ P < 0.001. H: Cells were treated as above and then immunocytochemical staining was conducted using fluorescently-labeled LPS (red) and anti-SRB1 antibody (green). Nuclei were counterstained by DAPI (blue). Scar bars, 20 μm.

Article Snippet: For lentiviral infection, RAW 264.7 cells were incubated with shSRB1 (Santa Cruz biotechnology, sc-44753-v) or non-targeting control (Santa Cruz biotechnology, sc-108080) (1 × 10 7 TU/ml) in RPMI supplemented with Polybrene (5 μg/ml) for 48 h. Transfected cells were sorted on day 5 post-infection by puromycin selection (4 μg/ml) followed by Western blotting to verify the gene silencing.

Techniques: Activation Assay, Inhibition, Incubation, Western Blot, Expressing, Control, Comparison, Enzyme-linked Immunosorbent Assay, Knockdown, Labeling, Staining, Flow Cytometry

Journal: Redox Biology

Article Title: S-nitrosylation of HINT1 in macrophages aggravates foam cell formation and atherosclerosis

doi: 10.1016/j.redox.2026.104063

Figure Lengend Snippet: S-nitrosylation of HINT1 aggravates ox-LDL induced foam cell formation through upregulating scavenger receptor A1 and CD36. (A) Representative images and quantification of oil red O staining in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). Scale bar: 50 μm. (B) Representative images and quantification of lipid droplet staining in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). Scale bar: 5 μm. (C) Total cholesterol level in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). (D) Representative images and quantification of Dil-ox-LDL uptake in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without Dil-ox-LDL (40 μg/mL) ( n = 6). Scale bar: 20 μm. (E) Cholesterol efflux to HDL was evaluated in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A and treated with or without ox-LDL (100 μg/mL) ( n = 6). (F) Western blot analysis of SR-A1, CD36 and LOX-1 in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). (G) Western blot analysis of SR-B1, ABCA1 and ABCG1 in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). (H) qPCR analysis of SR-A1, CD36 and LOX-1 in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). (I) qPCR analysis of SR-B1, ABCA1 and ABCG1 in HINT1 KO BMDMs transfected with Lenti-WT or Lenti-C84A followed by treatment with or without ox-LDL (100 μg/mL) ( n = 6). Data are presented as mean ± SEM. One-way ANOVA was used in A–C and E–I. Unpaired t -test was used in D.

Article Snippet: Antibodys against CD36 (18836-1-AP), SR-B1 (21277-1-AP), ABCA1 (26564-1-AP), ABCG1 (13578-1-AP), USF-2 (16614-1-AP), GAPDH (60004-1-lg), β-actin (66009-1-lg), and Tubulin (11224-1-AP) for western blotting, His-tag (66005-1-lg), Flag-tag (20543-1-AP), and HA-tag (51064-2-AP; 66006-2-lg) for western blotting and immunoprecipitation assay were obtained from Proteintech (Chicago, IL, USA); antibody against HINT1 (sc-271790), SR-A1 (sc-166184) for immunoprecipitation assay and immunofluorescence, CD36 (sc-7309) for immunofluorescence, USF2 (sc-293443) for immunoprecipitation assay were obtained from Santa Cruz Biotechnology (CA, USA).

Techniques: Staining, Transfection, Western Blot

Journal: Materials Today Bio

Article Title: Theranostic porphyrin nanoparticles identify atherosclerosis via multimodal imaging and elicit atheroprotective effects

doi: 10.1016/j.mtbio.2025.102202

Figure Lengend Snippet: Role of SR-B I and cholesterol efflux in the anti-inflammatory properties of Por-NPs in macrophages . iBMDMs were transfected with SR-BI siRNA (100 nM) to knockdown SR-BI. RT-qPCR measurement of (A) Il1b mRNA and (B) Ccl5 mRNA following incubation with discoidal Por-NPs and stimulation with LPS (10 ng/mL) (n = 3 biological replicates). iBMDMs were pre-incubated with PBS, methyl-β-cyclodextrin (MβCD, 4 % v/v), discoidal or CO-loaded Por-NPs (10 μg/mL) then stimulated with LPS (10 ng/mL) before RT-qPCR quantification of the mRNA levels of (C) Il1b and (D) Ccl5 . The nuclear fractions of (E) unstimulated and (F) LPS-stimulated iBMDMs were isolated and nuclear p65-NFκB protein levels (∼65 kDa) were assessed by Western blotting (n = 5–6 biological replicates). TATA binding protein (TBP, 37 kDa) was used as the nuclear protein loading control. Data expressed as Mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 by one-way ANOVA with post-hoc Tukey's multiple comparisons.

Article Snippet: iBMDMs were incubated with transfection reagent alone (Lipofectamine® RNAiMAX, 13778150, Thermofisher), scrambled control siRNA (sc-37007, Santa Cruz) or SR-BI siRNA (sc-44753, Santa Cruz) in optiMEM at 100 nM of siRNA for 6 h at 37 o C. Media was replaced and cells incubated for a total of 48 h iBMDMs were incubated with PBS or discoidal Por-NPs (10 μg/mL, R4F concentration, 24 h) then stimulated with LPS for 16 h for inflammatory gene assessments.

Techniques: Transfection, Knockdown, Quantitative RT-PCR, Incubation, Isolation, Western Blot, Binding Assay, Control

Journal: PLoS ONE

Article Title: Human Endothelial Progenitor Cells Internalize High-Density Lipoprotein

doi: 10.1371/journal.pone.0083189

Figure Lengend Snippet: A distinct green fluorescence is visible in the majority of outgrowing cells. Note that UEA-1 binding EPCs are negative for the SR-B1 receptor with exception of 1 cell which is weakly positive for UEA-1 (arrowhead). (Scale bar = 20 µm).

Article Snippet: In order to see whether or not EPCs express the scavenger receptor B1 (SR-B1) at their surface, a double labeling was performed using a purified monoclonal mouse IgG 1 antibody against the SR-B1 receptor (analogues: CL-A1, CD36 and LIMPII; clone 25/CLA-1; 250μg/ml) Becton Dickinson, Vienna, Austria) and a polyclonal goat-anti-mouse antibody, conjugated with Alexa Fluor® 488 (LifeTech Austria, Vienna, Austria) as 2 nd step reagent as well as with rhodamin-conjugated UEA-1 (10 mg/ml; Vector Laboratories, LTD, Peterborough, UK).

Techniques: Fluorescence, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: SR-B1 and CD36 express at the surface of Sf9 cells. Lysates from Sf9 cells infected with SR-B1– or CD36-expressing baculovirus at an MOI of 5 were collected every 24 h post-infection, following incubation with NHS-biotin as described in . Immunoblot analysis of 40 μl of biotinylated cell surface proteins ( top panels ) or 15 μg of whole-cell lysates ( bottom panels ) was performed and receptors were detected using an antibody directed against SR-B1 (approximately 82 kDa) or CD36 (approximately 88 kDa). Immunoblots are representative of four independent infections. SR-B1, scavenger receptor class B type 1; CD36, cluster of differentiation 36; MOI, multiplicity of infection; Sf9, Spodoptera frugiperda .

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Infection, Expressing, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: SR-B1–expressing Sf9 cells mediate DiI-HDL binding and DiI-lipid uptake from HDL. Sf9 cells were infected with high-titer baculovirus encoding SR-B1, CD36, or empty vector at an MOI of 5 and immediately plated in 12-well plates. At 72 h post-infection, cells were washed and incubated with 10 μg/ml DiI-HDL in media + 0.5% BSA for 90 min at 4 °C to assess binding ( A ) or at 27 °C to assess combined binding and uptake. After 90 min, cells were harvested in PBS/0.5% BSA. MFI was recorded by flow cytometry and DiI uptake ( B ) was calculated by subtracting the MFI at 4 °C from the MFI at 27 °C. Data were analyzed by one-way ANOVA and Dunnett’s multiple comparisons, presented as mean ± SD, n = 4 independent infections, with technical replicates performed in duplicate. ∗ p ≤ 0.05. BSA, bovine serum albumin; CD36, cluster of differentiation 36; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; HDL, high-density lipoprotein; MOI, multiplicity of infection; MST, microscale thermophoresis; Sf9, Spodoptera frugiperda ; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Infection, Plasmid Preparation, Incubation, Flow Cytometry, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: Sf9 cells expressing CD36 increase DiI-oxLDL binding and uptake. Sf9 cells were infected with high-titer baculovirus encoding SR-B1, CD36, or empty vector at an MOI of 5 and immediately plated in 12-well plates. At 72 h post-infection, cells were washed and incubated with 10 μg/ml DiI-oxLDL in media + 0.5% BSA for 90 min at 4 °C to assess binding ( A ) or at 27 °C to assess combined binding and uptake. After 90 min, cells were harvested in PBS/0.5% BSA. MFI was recorded by flow cytometry and DiI uptake ( B ) was calculated by subtracting the MFI at 4 °C from the MFI at 27 °C. Data were analyzed by one-way ANOVA and Dunnett’s multiple comparisons, presented as mean ± SD, n = 2 independent infections, with technical replicates performed in duplicate. ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01. BSA, bovine serum albumin; CD36, cluster of differentiation 36; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine; MFI, mean fluorescence intensity; MOI, multiplicity of infection; oxLDL, oxidized low-density lipoprotein; Sf9, Spodoptera frugiperda ; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Infection, Plasmid Preparation, Incubation, Flow Cytometry, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: SR-B1 and CD36 form higher order oligomers in Sf9 Cells. Cell lysates from empty vector (Vec), SR-B1-, or CD36-baculovirus-infected Sf9 cells were collected at 72 h post-infection. Lysates were sonicated and separated by nondenaturing perfluorooctanoic acid (PFO)-PAGE on 8% polyacrylamide gels. Immunoblot analysis utilized an antibody against SR-B1 ( A ) or CD36 ( B ) was used to detect the formation of monomers (M), dimers (D), and higher order oligomers (HO). Data are representative of immunoblots of lysates from three independent infections. CD36, cluster of differentiation 36; Sf9, Spodoptera frugiperda ; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Plasmid Preparation, Infection, Sonication, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: Schematic representation of scavenger receptor purification protocol. Sf9 cells were infected with baculovirus encoding SR-B1 or CD36, each flanked on the C terminus by a H10 histidine and FLAG tag. Lysates were solubilized with LMNG/CHS detergent (final concentration: 0.025% LMNG, 0.005% CHS) and tagged proteins were enriched by flow through a TALON cobalt affinity column. Tags and glycosylations can then be enzymatically removed. Final contaminants were removed by an additional flow through an affinity and desalting column. CD36, cluster of differentiation 36; CHS, cholesteryl hemisuccinate; LMNG, lauryl maltose neopentyl glycol; Sf9, Spodoptera frugiperda ; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Purification, Infection, FLAG-tag, Concentration Assay, Affinity Column

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: Purification protocol yields pure SR-B1 and CD36. Samples (∼15 μl) were collected at various points in the purification protocol and were separated by SDS-PAGE. Sample quality was verified by Coomassie blue ( A and B ) and immunoblot analysis ( C and D ). Size-exclusion chromatography for SR-B1 ( E ) or CD36 ( F ) was also performed. Blots are representative of four independent purifications. CD36, cluster of differentiation 36; FT, flow-through; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Purification, SDS Page, Western Blot, Size-exclusion Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: SR-B1 and CD36 remain stable up to 6 weeks post - purification. Purified SR-B1 and CD36 stability was measured up to 6 weeks post-purification by Prometheus NT.48. Protein unfolding as a function of increasing temperature was monitored by assessing changes in intrinsic tryptophan fluorescence at 330 and 350 nm at 20% power over a temperature gradient from 20 ˚C to 90 ˚C. Destabilization events for SR-B1 in detergent micelles occurred at 54.7 ˚C and 68.6 ˚C ( A ) and at 58.3 ˚C and 68.6 ˚C for CD36 ( B ). The destabilization temperatures were calculated by taking the ratio of the first derivative values of the sample fluorescence at 350 and 330 nm. Data represent two readings per construct per time point. Details of the thermal shift assay can be found in . CD36, cluster of differentiation 36; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Purification, Fluorescence, Construct, Thermal Shift Assay

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: Purified CD36 binds to oxLDL and HDL. MST was used to assess binding of Cy5-labeled SR-B1 to various ligands. Measurements were performed on the Monolith NT.115 BLUE/RED at 25 °C using 40% MST power and laser on/off times of 0 and 21 s. Each point is representative of a single point in a 16-point titration. Apparent K d values for glycosylated CD36 binding to oxLDL ( A , R 2 = 0.765) and HDL ( B , R 2 = 0.845) were calculated by nonlinear regression analysis, assuming one-site specific binding, of the normalized thermophoresis at increasing ligand concentrations, using GraphPad Prism. Curves are representative of at least two independent purifications, ligand preparations, and labelings. Further information about MST methods can be found in . CD36, cluster of differentiation 36; HDL, high-density lipoprotein; MST, microscale thermophoresis; oxLDL, oxidized low-density lipoprotein; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Purification, Binding Assay, Labeling, Titration, Microscale Thermophoresis

Journal: The Journal of Biological Chemistry

Article Title: Development and validation of a purification system for functional full-length human SR-B1 and CD36

doi: 10.1016/j.jbc.2023.105187

Figure Lengend Snippet: Glycosylation status of SR-B1 and CD36 does not impact ligand binding. Human full-length CD36 or SR-B1 were deglycosylated by PNGase F, as demonstrated by differences in migration by SDS-PAGE (insets). Proteins were subsequently labeled with Cy5 for MST experiments and performed as described in Figures 7 and . Apparent K d values for deglycosylated SR-B1 binding to HDL ( A , R 2 = 0.952) and deglycosylated CD36 binding to oxLDL ( B , R 2 = 0.799) were calculated by nonlinear regression analysis, assuming one-site specific binding, of the normalized thermophoresis at increasing ligand concentrations, using GraphPad Prism. MST curves of glycosylated proteins were calculated by nonlinear regression analysis, assuming one-site specific binding, of the normalized thermophoresis at increasing ligand concentrations, using GraphPad Prism. Curves are representative of at least two independent purifications, ligand preparations, and labelings. Further information about MST methods can be found in . CD36, cluster of differentiation 36; HDL, high-density lipoprotein; MST, microscale thermophoresis; oxLDL, oxidized low-density lipoprotein; SR-B1, scavenger receptor class B type 1.

Article Snippet: Purified SR-B1 or CD36 protein was labeled using the RED-NHS 2nd Generation Protein Labeling Kit (NanoTemper Technologies) according to manufacturer’s instructions.

Techniques: Ligand Binding Assay, Migration, SDS Page, Labeling, Binding Assay, Microscale Thermophoresis