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  • 90
    ATCC bac19796
    Summary of ORFs identified by significant homology (BLAST search) or GENEMARK prediction
    Bac19796, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bac19796/product/ATCC
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bac19796 - by Bioz Stars, 2024-04
    90/100 stars
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    91
    Santa Cruz Biotechnology rnf5 sirna
    <t>RNF5</t> interacts with SCAP on the ER membrane. A and B, anti-RNF5 IP experiments were performed using whole cell lysates prepared from (A) HuH-7 and (B) HepG2 cells. C and D, HEK293 cells were transfected with indicated combinations of plasmid and cultured for 48 h and then harvested for (C) anti-HA-SCAP IP and (D) anti–FLAG-RNF5 IP to examine RNF5-SCAP interaction. E, Insig-deficient SRD-15 cells were transfected with Myc-SCAP and/or FLAG-RNF5 expression plasmids and cultured for 48 h. The cells were then harvested for anti-FLAG-RNF5 IP to determine RNF5-SCAP interaction. F and G, HuH-7 cells were cultured in complete DMEM supplemented with 10% (v/v) FBS (F) or sterol-depleted medium supplemented with 5% LPDS (G) for 16 h and then harvested for subcellular fractionation. Sec61α and Golgi-97 represents a marker for the ER and the Golgi apparatus, respectively. H and I, HeLa cells were co-transfected with EGFP-SCAP and mCherry-RNF5 expression plasmids and cultured for 2 days in compete medium. Localization of EGFP-SCAP (green), mCherry-RNF5 (red), and ER (KDEL) or Golgi (GM130) markers (magenta) were then analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Enlarged images enclosed by dotted squares were shown in the right. Images for individual channels were shown in Fig. S2, A and B. Scale bar, 10 μm.
    Rnf5 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnf5 sirna/product/Santa Cruz Biotechnology
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnf5 sirna - by Bioz Stars, 2024-04
    91/100 stars
      Buy from Supplier

    86
    Millipore 95209 sog in water
    <t>RNF5</t> interacts with SCAP on the ER membrane. A and B, anti-RNF5 IP experiments were performed using whole cell lysates prepared from (A) HuH-7 and (B) HepG2 cells. C and D, HEK293 cells were transfected with indicated combinations of plasmid and cultured for 48 h and then harvested for (C) anti-HA-SCAP IP and (D) anti–FLAG-RNF5 IP to examine RNF5-SCAP interaction. E, Insig-deficient SRD-15 cells were transfected with Myc-SCAP and/or FLAG-RNF5 expression plasmids and cultured for 48 h. The cells were then harvested for anti-FLAG-RNF5 IP to determine RNF5-SCAP interaction. F and G, HuH-7 cells were cultured in complete DMEM supplemented with 10% (v/v) FBS (F) or sterol-depleted medium supplemented with 5% LPDS (G) for 16 h and then harvested for subcellular fractionation. Sec61α and Golgi-97 represents a marker for the ER and the Golgi apparatus, respectively. H and I, HeLa cells were co-transfected with EGFP-SCAP and mCherry-RNF5 expression plasmids and cultured for 2 days in compete medium. Localization of EGFP-SCAP (green), mCherry-RNF5 (red), and ER (KDEL) or Golgi (GM130) markers (magenta) were then analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Enlarged images enclosed by dotted squares were shown in the right. Images for individual channels were shown in Fig. S2, A and B. Scale bar, 10 μm.
    95209 Sog In Water, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/95209 sog in water/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    95209 sog in water - by Bioz Stars, 2024-04
    86/100 stars
      Buy from Supplier

    86
    Millipore 95209 s0g in water
    <t>RNF5</t> interacts with SCAP on the ER membrane. A and B, anti-RNF5 IP experiments were performed using whole cell lysates prepared from (A) HuH-7 and (B) HepG2 cells. C and D, HEK293 cells were transfected with indicated combinations of plasmid and cultured for 48 h and then harvested for (C) anti-HA-SCAP IP and (D) anti–FLAG-RNF5 IP to examine RNF5-SCAP interaction. E, Insig-deficient SRD-15 cells were transfected with Myc-SCAP and/or FLAG-RNF5 expression plasmids and cultured for 48 h. The cells were then harvested for anti-FLAG-RNF5 IP to determine RNF5-SCAP interaction. F and G, HuH-7 cells were cultured in complete DMEM supplemented with 10% (v/v) FBS (F) or sterol-depleted medium supplemented with 5% LPDS (G) for 16 h and then harvested for subcellular fractionation. Sec61α and Golgi-97 represents a marker for the ER and the Golgi apparatus, respectively. H and I, HeLa cells were co-transfected with EGFP-SCAP and mCherry-RNF5 expression plasmids and cultured for 2 days in compete medium. Localization of EGFP-SCAP (green), mCherry-RNF5 (red), and ER (KDEL) or Golgi (GM130) markers (magenta) were then analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Enlarged images enclosed by dotted squares were shown in the right. Images for individual channels were shown in Fig. S2, A and B. Scale bar, 10 μm.
    95209 S0g In Water, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/95209 s0g in water/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    95209 s0g in water - by Bioz Stars, 2024-04
    86/100 stars
      Buy from Supplier


    Image Search Results


    Summary of ORFs identified by significant homology (BLAST search) or GENEMARK prediction

    Journal:

    Article Title: Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

    doi: 10.1128/JB.186.1.212-225.2004

    Figure Lengend Snippet: Summary of ORFs identified by significant homology (BLAST search) or GENEMARK prediction

    Article Snippet: 36 , 215 , 33754-34398 , Putative membrane protein ( Corynebacterium efficiens ), COG1651, related to thiol-disulfide oxidoreductase BdbD ( B. cereus ATCC 14579), pfam01323 , {"type":"entrez-protein","attrs":{"text":"BAC19796","term_id":"23494833","term_text":"BAC19796"}} BAC19796 , 95/209 (45%) , 8e−40.

    Techniques: Sequencing, Modification, Plasmid Preparation, Binding Assay

    RNF5 interacts with SCAP on the ER membrane. A and B, anti-RNF5 IP experiments were performed using whole cell lysates prepared from (A) HuH-7 and (B) HepG2 cells. C and D, HEK293 cells were transfected with indicated combinations of plasmid and cultured for 48 h and then harvested for (C) anti-HA-SCAP IP and (D) anti–FLAG-RNF5 IP to examine RNF5-SCAP interaction. E, Insig-deficient SRD-15 cells were transfected with Myc-SCAP and/or FLAG-RNF5 expression plasmids and cultured for 48 h. The cells were then harvested for anti-FLAG-RNF5 IP to determine RNF5-SCAP interaction. F and G, HuH-7 cells were cultured in complete DMEM supplemented with 10% (v/v) FBS (F) or sterol-depleted medium supplemented with 5% LPDS (G) for 16 h and then harvested for subcellular fractionation. Sec61α and Golgi-97 represents a marker for the ER and the Golgi apparatus, respectively. H and I, HeLa cells were co-transfected with EGFP-SCAP and mCherry-RNF5 expression plasmids and cultured for 2 days in compete medium. Localization of EGFP-SCAP (green), mCherry-RNF5 (red), and ER (KDEL) or Golgi (GM130) markers (magenta) were then analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Enlarged images enclosed by dotted squares were shown in the right. Images for individual channels were shown in Fig. S2, A and B. Scale bar, 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

    doi: 10.1074/jbc.RA119.011849

    Figure Lengend Snippet: RNF5 interacts with SCAP on the ER membrane. A and B, anti-RNF5 IP experiments were performed using whole cell lysates prepared from (A) HuH-7 and (B) HepG2 cells. C and D, HEK293 cells were transfected with indicated combinations of plasmid and cultured for 48 h and then harvested for (C) anti-HA-SCAP IP and (D) anti–FLAG-RNF5 IP to examine RNF5-SCAP interaction. E, Insig-deficient SRD-15 cells were transfected with Myc-SCAP and/or FLAG-RNF5 expression plasmids and cultured for 48 h. The cells were then harvested for anti-FLAG-RNF5 IP to determine RNF5-SCAP interaction. F and G, HuH-7 cells were cultured in complete DMEM supplemented with 10% (v/v) FBS (F) or sterol-depleted medium supplemented with 5% LPDS (G) for 16 h and then harvested for subcellular fractionation. Sec61α and Golgi-97 represents a marker for the ER and the Golgi apparatus, respectively. H and I, HeLa cells were co-transfected with EGFP-SCAP and mCherry-RNF5 expression plasmids and cultured for 2 days in compete medium. Localization of EGFP-SCAP (green), mCherry-RNF5 (red), and ER (KDEL) or Golgi (GM130) markers (magenta) were then analyzed by confocal microscopy. Nuclei were stained with DAPI (blue). Enlarged images enclosed by dotted squares were shown in the right. Images for individual channels were shown in Fig. S2, A and B. Scale bar, 10 μm.

    Article Snippet: Stealth RNAi siRNA Negative Control, Med GC (code: 12935300) and RNF5 Stealth siRNA were purchased from Thermo Fisher Scientific (assay ID: HSS155076, HSS155077, HSS155078), and control siRNA-A and RNF5 siRNA were purchased from Santa Cruz Biotechnology (codes: sc-37007 and sc-95209, respectively).

    Techniques: Transfection, Plasmid Preparation, Cell Culture, Expressing, Fractionation, Marker, Confocal Microscopy, Staining

    RNF5 knockdown inhibits SREBP2 activation to block cholesterol biosynthesis. A, HuH-7 cells were transfected with control (sc-37007) or RNF5-specific siRNA (sc-95209) or transfected with pcDNA3.1 empty vector or pcDNA-RNF5 expression plasmid. The cells were cultured for 32 h and then switched to sterol-depleted medium and cultured for another 16 h and then harvested for immunoblot analysis. B, HuH-7 cells were transfected with a control siRNA (assay ID: 12935300) or three RNF5-specific siRNA targeting different locations (assay ID: HSS155076, HSS155077, HSS155078). The cells were cultured for 32 h and then switch to sterol-depleted medium supplemented with 1 μg/ml 25-HC or with equal volume of ethanol and cultured for another 16 h and then harvested for immunoblot analysis. In A and B, arrows indicate RNF5 protein bands, whereas asterisks indicate nonspecific protein bands. C, HEK293 cells were reverse transfected with His-SREBP2 expression plasmid and cultured for 24 h. The cells were then transfected with siRNA as described in (A) and then switched to fresh medium as described in (B). The cells were then harvested for immunoblot analysis. A–C, the precursor (P) form and the nuclear (N) form of SREBP2 was detected by anti-SREBP2 (RS004) antibody that recognizes the N-terminal domain of SREBP2. D, HuH-7 cells were transfected with siRNA and cultured as described in (A). The cells were then harvested for total RNA extraction followed by reverse transcription and quantitative PCR. E, HuH-7 cells were transfected with siRNA as described in (A) and switched to fresh medium as described in (B). The cells were then harvested for immunoblot analysis. F, HuH-7 cells were treated as described in (D). The cells were then harvested for quantification of cellular cholesterol. G, HuH-7 cells were transfected with siRNA and cultured as described in (A). A group of cells was mock transfected and then cultured in sterol-depleted medium containing 1 μg/ml 25-HC. De novo cholesterol biosynthesis was determined as described under “Experimental procedures.” The Bar chart shows the signal of [14C]-labeled cholesterol normalized by cellular protein level. The image showing cholesterol bands shows the [14C]-cholesterol bands visualized by TLC. The unit of radiolabeled signal is presented as PSL-BG, which stands for photostimulated luminescence minus background. Data shown in D and F were pooled from three independent experiments performed in triplicate (n = 9), whereas data shown in (G) were pooled from two independent experiments performed in triplicate (n = 6). In D, F, and G, data are presented as mean ± S.D. Asterisks indicate difference between groups were significant as determined by two-tailed unpaired Student's t-tests (p < 0.05).

    Journal: The Journal of Biological Chemistry

    Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

    doi: 10.1074/jbc.RA119.011849

    Figure Lengend Snippet: RNF5 knockdown inhibits SREBP2 activation to block cholesterol biosynthesis. A, HuH-7 cells were transfected with control (sc-37007) or RNF5-specific siRNA (sc-95209) or transfected with pcDNA3.1 empty vector or pcDNA-RNF5 expression plasmid. The cells were cultured for 32 h and then switched to sterol-depleted medium and cultured for another 16 h and then harvested for immunoblot analysis. B, HuH-7 cells were transfected with a control siRNA (assay ID: 12935300) or three RNF5-specific siRNA targeting different locations (assay ID: HSS155076, HSS155077, HSS155078). The cells were cultured for 32 h and then switch to sterol-depleted medium supplemented with 1 μg/ml 25-HC or with equal volume of ethanol and cultured for another 16 h and then harvested for immunoblot analysis. In A and B, arrows indicate RNF5 protein bands, whereas asterisks indicate nonspecific protein bands. C, HEK293 cells were reverse transfected with His-SREBP2 expression plasmid and cultured for 24 h. The cells were then transfected with siRNA as described in (A) and then switched to fresh medium as described in (B). The cells were then harvested for immunoblot analysis. A–C, the precursor (P) form and the nuclear (N) form of SREBP2 was detected by anti-SREBP2 (RS004) antibody that recognizes the N-terminal domain of SREBP2. D, HuH-7 cells were transfected with siRNA and cultured as described in (A). The cells were then harvested for total RNA extraction followed by reverse transcription and quantitative PCR. E, HuH-7 cells were transfected with siRNA as described in (A) and switched to fresh medium as described in (B). The cells were then harvested for immunoblot analysis. F, HuH-7 cells were treated as described in (D). The cells were then harvested for quantification of cellular cholesterol. G, HuH-7 cells were transfected with siRNA and cultured as described in (A). A group of cells was mock transfected and then cultured in sterol-depleted medium containing 1 μg/ml 25-HC. De novo cholesterol biosynthesis was determined as described under “Experimental procedures.” The Bar chart shows the signal of [14C]-labeled cholesterol normalized by cellular protein level. The image showing cholesterol bands shows the [14C]-cholesterol bands visualized by TLC. The unit of radiolabeled signal is presented as PSL-BG, which stands for photostimulated luminescence minus background. Data shown in D and F were pooled from three independent experiments performed in triplicate (n = 9), whereas data shown in (G) were pooled from two independent experiments performed in triplicate (n = 6). In D, F, and G, data are presented as mean ± S.D. Asterisks indicate difference between groups were significant as determined by two-tailed unpaired Student's t-tests (p < 0.05).

    Article Snippet: Stealth RNAi siRNA Negative Control, Med GC (code: 12935300) and RNF5 Stealth siRNA were purchased from Thermo Fisher Scientific (assay ID: HSS155076, HSS155077, HSS155078), and control siRNA-A and RNF5 siRNA were purchased from Santa Cruz Biotechnology (codes: sc-37007 and sc-95209, respectively).

    Techniques: Activation Assay, Blocking Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction, Labeling, Two Tailed Test

    RNF5 mediates Lys-29–linked polyubiquitination of SCAP. A, SRD-13A cells were seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were then switched to Opti-MEM and transfected with 0.5 μg/well HA-ubiquitin (Ub), 0.5 μg/well Myc-SCAP, and/or 0.5 or 1.0 μg/well RNF5 expression plasmid and incubated for 2 h. The total amount of transfected plasmid was adjusted to 2 μg/well using pcDNA3.1 empty vector. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 20 h. The cells were then switched to fresh medium supplemented with 5% LPDS and 10 μm MG132 and cultured for another 2 h. The cells were then harvested for anti-Myc-SCAP IP. B, SRD-13A cells were transfected with 1.0 μg/well HA-Ub, 0.5 μg/well Myc-SCAP and/or 0.5 μg/well WT or mutant RNF5 expression plasmid and then cultured for IP experiment as described in (A). C and D, SRD-13A cells were transfected with 0.5 μg/well Myc-SCAP and WT or 1.0 μg/well mutant HA-Ub in the presence or absence of 0.5 μg/well RNF5 expression plasmid and then cultured for IP experiment as described in (A).

    Journal: The Journal of Biological Chemistry

    Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

    doi: 10.1074/jbc.RA119.011849

    Figure Lengend Snippet: RNF5 mediates Lys-29–linked polyubiquitination of SCAP. A, SRD-13A cells were seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were then switched to Opti-MEM and transfected with 0.5 μg/well HA-ubiquitin (Ub), 0.5 μg/well Myc-SCAP, and/or 0.5 or 1.0 μg/well RNF5 expression plasmid and incubated for 2 h. The total amount of transfected plasmid was adjusted to 2 μg/well using pcDNA3.1 empty vector. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 20 h. The cells were then switched to fresh medium supplemented with 5% LPDS and 10 μm MG132 and cultured for another 2 h. The cells were then harvested for anti-Myc-SCAP IP. B, SRD-13A cells were transfected with 1.0 μg/well HA-Ub, 0.5 μg/well Myc-SCAP and/or 0.5 μg/well WT or mutant RNF5 expression plasmid and then cultured for IP experiment as described in (A). C and D, SRD-13A cells were transfected with 0.5 μg/well Myc-SCAP and WT or 1.0 μg/well mutant HA-Ub in the presence or absence of 0.5 μg/well RNF5 expression plasmid and then cultured for IP experiment as described in (A).

    Article Snippet: Stealth RNAi siRNA Negative Control, Med GC (code: 12935300) and RNF5 Stealth siRNA were purchased from Thermo Fisher Scientific (assay ID: HSS155076, HSS155077, HSS155078), and control siRNA-A and RNF5 siRNA were purchased from Santa Cruz Biotechnology (codes: sc-37007 and sc-95209, respectively).

    Techniques: Cell Culture, Transfection, Expressing, Plasmid Preparation, Incubation, Mutagenesis

    RNF5 ubiquitinates lysine Lys-305 located in cytosolic loop 2 of SCAP. A, SRD-13A cells were seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were then switched to Opti-MEM and transfected with 0.5 μg/well HA-ubiquitin (Ub) and 1.0 μg/well WT or mutant Myc-SCAP in the presence or absence of 0.5 μg/well RNF5 expression plasmid and incubated for 2 h. The total amount of transfected plasmid was adjusted to 2 μg/well using pcDNA3.1 empty vector. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 20 h and then switched to fresh medium supplemented with 5% LPDS and 10 μm MG132 and cultured for another 2 h. The cells were then harvested for anti-Myc-SCAP IP. B, schematic presentation of the FLAG-SCAP plasmids used in (C). FL indicates full-length of SCAP, whereas plasmid no.1 to 4 are truncated from either C-terminal or N-terminal end of SCAP. TM indicates transmembrane domain, whereas L indicates loops pointed to the cytosol. Lysine residue Lys-305 on loop 2 is highlighted in red. C, SRD-13A cells were seeded as described in (A) and transfected with 2 μg/well empty vector or WT or truncated FLAG-SCAP plasmids in Opti-MEM and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h. The cells were then harvested for anti-FLAG-SCAP IP. D, alignment of RNF5-binding region of SCAP and STING (14). Gray boxes indicate the amino acid sequences of transmembrane domains TM2 and TM3. White box indicates the amino acid sequence of cytosolic loop 2. The lysine residues that were ubiquitinated by RNF5 in SCAP and STING are highlighted in red.

    Journal: The Journal of Biological Chemistry

    Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

    doi: 10.1074/jbc.RA119.011849

    Figure Lengend Snippet: RNF5 ubiquitinates lysine Lys-305 located in cytosolic loop 2 of SCAP. A, SRD-13A cells were seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were then switched to Opti-MEM and transfected with 0.5 μg/well HA-ubiquitin (Ub) and 1.0 μg/well WT or mutant Myc-SCAP in the presence or absence of 0.5 μg/well RNF5 expression plasmid and incubated for 2 h. The total amount of transfected plasmid was adjusted to 2 μg/well using pcDNA3.1 empty vector. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 20 h and then switched to fresh medium supplemented with 5% LPDS and 10 μm MG132 and cultured for another 2 h. The cells were then harvested for anti-Myc-SCAP IP. B, schematic presentation of the FLAG-SCAP plasmids used in (C). FL indicates full-length of SCAP, whereas plasmid no.1 to 4 are truncated from either C-terminal or N-terminal end of SCAP. TM indicates transmembrane domain, whereas L indicates loops pointed to the cytosol. Lysine residue Lys-305 on loop 2 is highlighted in red. C, SRD-13A cells were seeded as described in (A) and transfected with 2 μg/well empty vector or WT or truncated FLAG-SCAP plasmids in Opti-MEM and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h. The cells were then harvested for anti-FLAG-SCAP IP. D, alignment of RNF5-binding region of SCAP and STING (14). Gray boxes indicate the amino acid sequences of transmembrane domains TM2 and TM3. White box indicates the amino acid sequence of cytosolic loop 2. The lysine residues that were ubiquitinated by RNF5 in SCAP and STING are highlighted in red.

    Article Snippet: Stealth RNAi siRNA Negative Control, Med GC (code: 12935300) and RNF5 Stealth siRNA were purchased from Thermo Fisher Scientific (assay ID: HSS155076, HSS155077, HSS155078), and control siRNA-A and RNF5 siRNA were purchased from Santa Cruz Biotechnology (codes: sc-37007 and sc-95209, respectively).

    Techniques: Cell Culture, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Binding Assay, Sequencing

    RNF5-dependent polyubiquitination enhances luminal loop 1 and loop 7 interaction of SCAP. A, SRD-13A was seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were switched to Opti-MEM and transfected with 0.5 μg/well FLAG-SCAP(amino acids 2–472) (L1) and/or 0.5 μg/well Myc-SCAP(amino acids 496–1279) (L7) and 0.5, 0.75, 1.0 μg/well RNF5 expression plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. B, SRD-13A were cultured as described in (A) and transfected with 1.0 μg/well HA-ubiquitin and/or 0.5 μg/well FLAG-SCAP or FLAG-L1 in the presence or absence of 0.5 μg/well RNF5 expression plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. C, SRD-13A were as described in (A) and transfected with 1.0 μg/well Myc-L7 and/or 1.0 μg/well FLAG-L1 or FLAG-L1 K305R mutant plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. D, schematic illustration of the proposed mechanism. RNF5 induces polyubiquitination of SCAP at Lys-305, promoting loop 1–loop 7 interaction, which is a crucial event for SREBP-mediated cholesterol biosynthetic pathway.

    Journal: The Journal of Biological Chemistry

    Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

    doi: 10.1074/jbc.RA119.011849

    Figure Lengend Snippet: RNF5-dependent polyubiquitination enhances luminal loop 1 and loop 7 interaction of SCAP. A, SRD-13A was seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were switched to Opti-MEM and transfected with 0.5 μg/well FLAG-SCAP(amino acids 2–472) (L1) and/or 0.5 μg/well Myc-SCAP(amino acids 496–1279) (L7) and 0.5, 0.75, 1.0 μg/well RNF5 expression plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. B, SRD-13A were cultured as described in (A) and transfected with 1.0 μg/well HA-ubiquitin and/or 0.5 μg/well FLAG-SCAP or FLAG-L1 in the presence or absence of 0.5 μg/well RNF5 expression plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. C, SRD-13A were as described in (A) and transfected with 1.0 μg/well Myc-L7 and/or 1.0 μg/well FLAG-L1 or FLAG-L1 K305R mutant plasmids and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h and then harvested for anti-Myc IP. D, schematic illustration of the proposed mechanism. RNF5 induces polyubiquitination of SCAP at Lys-305, promoting loop 1–loop 7 interaction, which is a crucial event for SREBP-mediated cholesterol biosynthetic pathway.

    Article Snippet: Stealth RNAi siRNA Negative Control, Med GC (code: 12935300) and RNF5 Stealth siRNA were purchased from Thermo Fisher Scientific (assay ID: HSS155076, HSS155077, HSS155078), and control siRNA-A and RNF5 siRNA were purchased from Santa Cruz Biotechnology (codes: sc-37007 and sc-95209, respectively).

    Techniques: Cell Culture, Transfection, Expressing, Incubation, Mutagenesis