atp binding cassette  (Proteintech)

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 promoter activity in CD8+ T cells from the thymus and the spleen. Spleens and thymi were dissected from threeABCF1+/- (top panels) and three of their wild-type littermate controls (bottom panels). Cells were labelled with the αnti-CD8 antibody and αnti-CD4 antibody. We depict the staining of two ABCF1+/- and one wild-type littermate controls. Data is representative of two separate experiments.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Activity Assay, Staining

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 promoter activity in CD8+ T cells from the thymus and the spleen. Spleens and thymi were dissected out of three ABCF1+/- (top panels) and three of their wild-type littermate controls (bottom panels). Cells were labelled with the PE-αCD8 antibody and then stained with fluorescein by heat shock. Data is representative of two separate experiments.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Activity Assay, Staining

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 +/- mice produce fewer IFN γ positive CD8+ T lymphocytes after infection with VSV. ABCF1+/- splenocytes showed fewer IFNγ positive CD8+ T lymphocytes. Figure shown represents the results of one experiment, which was later repeated three times with similar results.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Infection

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1+/- CTLs were as efficient as ABCF1 +/+ at killing of chromium. loaded VSV specific target lymphocytes. CTL activity was measured by the release of 51 Cr into the supernatant. Figure shown represents the results of one experiment, which was later repeated three times with similar results.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 is essential for the production of ISG (Interferon-Stimulated Gene) specific cytokines expression of INF- α and β after Poly(I:C) stimulation. A bar graph illustrating the fold change in the levels of INF-α and β cytokines detected in the cell culture supernatants. These findings provide insights into the impact of Abcf1 -specific siRNA treatment, both with and without Poly(I:C), on the secretion of cytokines and chemokines as well as the activation of key signaling pathways and transcription factors within BMDMs. Expression levels of the protein was assessed by western blotting analysis.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Expressing, Cell Culture, Activation Assay, Protein-Protein interactions, Western Blot

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 plays a role in regulating the phosphorylation levels of NF- κ B p65 and IRF3. A) Cytoplasmic and nuclear fractions obtained from BMDMs under different conditions were subjected to western blot analysis to assess the levels of transcription factors associated with Poly(I:C) stimulation confirming our hypothesis; Western Blot band sizes are as follows: anti- p65 (65 kD); anti p-p65 (65 kD); anti IRF3 (47 kD); anti p-IRF3 (36 kD); anti ABCF1 (110 kD); anti GAPDH (36 kD); anti histone H3 (15 kD); Dimerized-IRF3 (73 kD); IRF3 (36 kD) B) Whole-cell lysates were separated using native PAGE gel electrophoresis, and the analysis focused on examining the dimerization of IRF3, a transcription factor. Expression levels of the protein was assessed by western blotting analysis. Fold change calculations, as specified in the materials and methods section, were employed to quantify changes in the data. The bar graphs displayed in the figures represent the mean values along with standard deviations. The data presented here is representative of three separate experiments.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Phospho-proteomics, Western Blot, Clear Native PAGE, Nucleic Acid Electrophoresis, Expressing

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 Parallels OAS1a Expression and Controls 2-5A Activity A) The immunoprecipitates were immunoblotted with anti-ABCF1 and anti-OAS1a antibodies. B) ABCF1 was over-expressed (Over Exp.) in BMDMs, an equal amount of whole cell lysates was immunoprecipitated with anti-ABCF1 antibody and the immunoprecipitates were detected with anti-ABCF1 and anti-OAS1a antibodies using western blots. C) Protein levels of OAS1a, ABCE1 and RNase L were analyzed from whole cell lysates from Abcf1 siRNA-treated BMDMs in presence or absence of Poly(I:C). Expression levels of the protein was assessed by western blotting analysis. D) Increased pyrophosphate production (a readout of 2-5A activity) was measured in scrambled and Abcf1 siRNA treated BMDMs in presence and absence of recombinant OAS1. E) Total RNA was extracted from Abcf1 siRNA treated BMDMs in presence or absence of Poly(I:C) and was run on a Agilent 2100 Bioanalyzer. 28S and 18S RNA have been shown and RIN score was calculated. Bars indicate the mean ±standard deviation. The data is representative of 3 separate experiments. The expression of ABCF1 follows a pattern that parallels the expression of OAS1a, and it also exerts control over the activity of 2-5A. This suggests a regulatory relationship where ABCF1’s expression correlates with OAS1a expression and influences the activity of 2-5A.

Article Snippet: For all co-immunoprecipitation (Co-IP) samples, the sample buffer was formulated without dithiothreitol (DTT), unless explicitly stated otherwise (Anti ABCF1 antibody for co- immunoprecipitation was sourced from Proteintech; catalog 13950-1-AP).

Techniques: Expressing, Activity Assay, Immunoprecipitation, Western Blot, Recombinant, Standard Deviation, Control