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peripheral blood cells  (ATCC)
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ATCC peripheral blood cells
Peripheral Blood Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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primary human cd14 monocytes  (ATCC)
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ATCC primary human cd14 monocytes
(A) FLAG immunoprecipitation was performed from lysates of HEK293T cells expressing FLAG-RNF213 or FLAG-eGFP in combination with HA-ISG15(AA) and the ISGylation machinery (E1, E2, E3). A smear of ISGylated co-immunoprecipitated proteins was detected with FLAG-RNF213, but not with FLAG-eGFP or when non-conjugatable HA-ISG15AA was used. (B) . THP-1 or primary human monocytes <t>(CD14+)</t> cells were cultured in the presence of 10 mM BSA-conjugated oleic acid and either treated with 10 ng/mL interferon-β for 8 h or left untreated. Lipid droplets (LDs)-enriched fractions were isolated by ultracentrifugation floatation assay on a sucrose step-gradient. Immunoblots against RNF213 and ISG15 revealed an interferon-induced upregulation of both proteins on LDs and a smear of ISGylated proteins associated with LDs. Immunoblots against PLIN1, PLIN2, ATGL and GAPDH confirmed LD isolation and equal protein loading in the lysate and LD-enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded). (C) Similarly, LDs were isolated from THP1 cells after knockdown of RNF213 by siRNA treatment for 48h or using a non-targeting scrambled siRNA (siScramble) as control. Immunoblotting against ISG15 revealed a smear of ISGylated proteins associated with LDs only when RNF213 was present. Immunoblotting against RNF213 confirmed knockdown of RNF213, while PLIN1, PLIN2, ATGL and GAPDH validated LD isolation and equal protein loading in the lysate and the LD- enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded).
Primary Human Cd14 Monocytes, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/primary+peripheral+blood+cd14+monocytes/Primary+Peripheral+Blood+CD14%2B+Monocytes%2C+Normal%2C+Human/bio_rxiv__2021__06__03__446796-311-22-26
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(A) FLAG immunoprecipitation was performed from lysates of HEK293T cells expressing FLAG-RNF213 or FLAG-eGFP in combination with HA-ISG15(AA) and the ISGylation machinery (E1, E2, E3). A smear of ISGylated co-immunoprecipitated proteins was detected with FLAG-RNF213, but not with FLAG-eGFP or when non-conjugatable HA-ISG15AA was used. (B) . THP-1 or primary human monocytes (CD14+) cells were cultured in the presence of 10 mM BSA-conjugated oleic acid and either treated with 10 ng/mL interferon-β for 8 h or left untreated. Lipid droplets (LDs)-enriched fractions were isolated by ultracentrifugation floatation assay on a sucrose step-gradient. Immunoblots against RNF213 and ISG15 revealed an interferon-induced upregulation of both proteins on LDs and a smear of ISGylated proteins associated with LDs. Immunoblots against PLIN1, PLIN2, ATGL and GAPDH confirmed LD isolation and equal protein loading in the lysate and LD-enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded). (C) Similarly, LDs were isolated from THP1 cells after knockdown of RNF213 by siRNA treatment for 48h or using a non-targeting scrambled siRNA (siScramble) as control. Immunoblotting against ISG15 revealed a smear of ISGylated proteins associated with LDs only when RNF213 was present. Immunoblotting against RNF213 confirmed knockdown of RNF213, while PLIN1, PLIN2, ATGL and GAPDH validated LD isolation and equal protein loading in the lysate and the LD- enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded).

Journal: bioRxiv

Article Title: Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

doi: 10.1101/2021.06.03.446796

Figure Lengend Snippet: (A) FLAG immunoprecipitation was performed from lysates of HEK293T cells expressing FLAG-RNF213 or FLAG-eGFP in combination with HA-ISG15(AA) and the ISGylation machinery (E1, E2, E3). A smear of ISGylated co-immunoprecipitated proteins was detected with FLAG-RNF213, but not with FLAG-eGFP or when non-conjugatable HA-ISG15AA was used. (B) . THP-1 or primary human monocytes (CD14+) cells were cultured in the presence of 10 mM BSA-conjugated oleic acid and either treated with 10 ng/mL interferon-β for 8 h or left untreated. Lipid droplets (LDs)-enriched fractions were isolated by ultracentrifugation floatation assay on a sucrose step-gradient. Immunoblots against RNF213 and ISG15 revealed an interferon-induced upregulation of both proteins on LDs and a smear of ISGylated proteins associated with LDs. Immunoblots against PLIN1, PLIN2, ATGL and GAPDH confirmed LD isolation and equal protein loading in the lysate and LD-enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded). (C) Similarly, LDs were isolated from THP1 cells after knockdown of RNF213 by siRNA treatment for 48h or using a non-targeting scrambled siRNA (siScramble) as control. Immunoblotting against ISG15 revealed a smear of ISGylated proteins associated with LDs only when RNF213 was present. Immunoblotting against RNF213 confirmed knockdown of RNF213, while PLIN1, PLIN2, ATGL and GAPDH validated LD isolation and equal protein loading in the lysate and the LD- enriched fraction (1/20 th of the lysate and all of the LD-enriched material was loaded).

Article Snippet: Hek293T cells originate from (Lin et al., 2014) while HeLa cells (ATCC® CCL-2TM), A549 cells (ATCC® CCL-185TM) THP-1 cells (ATCC® TIB-202TM) and primary human CD14+ monocytes (ATCC® PCS- 800-010 TM ) were purchased from ATCC.

Techniques: Immunoprecipitation, Expressing, Cell Culture, Isolation, Western Blot, Knockdown, Control