rabbit polyclonal anti osbp  (Proteintech)

Journal: Proceedings of the National Academy of Sciences

Article Title: Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages

doi: 10.1073/pnas.2406492121

Figure Lengend Snippet: Fig. 2. Osbp mediates macrophage inflammatory responses to 25HC. (A) Secretion of Il6 measured by ELISA in BMDMs left untreated or treated with the indicated Osbp ligands and then stimulated with LPS or PAM3 for 18 h. (B) BMDMs were left untreated or treated with 25HC, itraconazole, or OSW-1 then were left unstimulated or stimulated with LPS and their transcriptomes analyzed by RNA-seq. Top panels: Comparison of the effect of the indicated Osbp ligands on the expression of genes whose expression is altered by at least two-fold (FDR < 0.01; avg. log2(counts-per-million reads) > 2 across all conditions) by treatment with at least one of the indicated Osbp ligands. Bottom panels: The core LPS response was defined as the 637 genes whose expression at 6 h was altered by at least 10-fold (FDR < 0.01; avg. log2(CPM reads) > 2 across all conditions). The plots compare the effect of itraconazole or OSW-1 to that of 25HC on the expression at 18 h following stimulation of core response genes whose expression at 18 h was altered by at least two-fold by either of the indicated treatments on each plot. (C) Percentage of unedited sequences remaining in the indicated cell pools as measured by TIDE sequencing (29). (D) Western blot for Osbp in macrophages differentiated from Osbp g1, g5, and g6 pools. (E) Il6 protein level 18 h following LPS stimulation of macrophages derived from the indicated CIM lines and treated with the indicated compounds. Bars/points indicate the mean of three biological replicates; error bars indicate SEM (A and E). The blue line indicates best fit and r = Pearson correlation (B). Similar results were obtained in 3 (A) or 2 (E) independent experiments.

Article Snippet: 121 No. 33 e2406492121 https://doi.org/10.1073/pnas.2406492121 7 of 8 After blocking with 5% milk, membranes were probed with the relevant primary antibodies (Cleaved Il1b – E7V2A (Cell Signaling #63124), Osbp Polyclonal (Proteintech #11096- 1- AP)) followed by detection with an HRP- conjugated secondary antibody (Anti- mouse IgG- HRP (Cell Signaling #7076), Anti- rabbit IgG HRP (Cell Signaling #7074)).

Techniques: Enzyme-linked Immunosorbent Assay, RNA Sequencing, Comparison, Expressing, Sequencing, Western Blot, Derivative Assay

Journal: Redox Biology

Article Title: The CH24H metabolite, 24HC, blocks viral entry by disrupting intracellular cholesterol homeostasis

doi: 10.1016/j.redox.2023.102769

Figure Lengend Snippet: 24HC sequesters the viral particles penetrating the MVB/LE and blocks the OSBP-VAPA interaction. A. N2a cells were pretreated with 5 μM 24HC for 12 h, and then infected with VSV (MOI = 10) for 3 h. Cells were analyzed for the co-localization between VSV nucleoprotein (VSV–N) and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. B. N2a cells were pretreated with 5 μM 24HC for 12 h, and then infected with RABV (MOI = 10) for 3 h. Cells were analyzed for the co-localization between RABV nucleoprotein (RABV-N) and Rab7. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. C. Co-immunoprecipitation (Co-IP) was performed to detect the effect of 24HC and 25HC treatment on the interaction between OSBP and VAPA. N2a cells treated with ETOH, 24HC (5 μM), or 25HC (5 μM) for 36 h were harvested and lysed, and then pulled down using OSBP antibody as a bait. The precipitated proteins were analyzed by WB. Grey value comparison was calculated using ImageJ software. D. N2a cells were transfected with expression plasmid pCAGGS-FLAG-VAPA (named FLAG-VAPA) for 12 h and then treated with different concentrations of 24HC for 24 h. FLAG antibody was used as bait and the precipitated proteins were analyzed by WB. E. N2a cells were transfected with FLAG-VAPA for 12 h and then treated with 5 μM 24HC for 24 h, cells were fixed and stained for OSBP, FLAG-VAPA, and DAPI. The cells were then observed by confocal microscopy. White boxes indicate the magnified sections of the images. Scale bar, 10 μm. F. N2a cells were transfected with the expression plasmid pCAGGS-HA-OSBP (named HA-OSBP). After 12 h post-transfection, 5 μM 24HC was added to N2a cell culture media for 24 h. Cells were then fixed and stained with HA-OSBP and DAPI, and the Z-stack of confocal microscopy was used to acquire multi-layer images. The multi-layer images were imported into the ZEN software (version 2.3) to obtain a rendered 3D volume image. Scale bar = 10 μm. The Western blot data are representative of at least two independent experiments.

Article Snippet: The pAbs against OSBP (11096-1-AP) and VAPA (15275-1-AP) were purchased from Proteintech (Chicago, USA).

Techniques: Infection, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison, Software, Transfection, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Cell Culture, Western Blot