a microtome  (Thermo Fisher)


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    Structured Review

    Thermo Fisher a microtome
    A Microtome, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 87 stars, based on 3 article reviews
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    Modification:

    Article Title: Gliclazide alone or in combination with atorvastatin ameliorated reproductive damage in streptozotocin-induced type 2 diabetic male rats
    Article Snippet: Right testis were immediately fixed in Davidson’s modified fixative and kept at 4 °C for 24 h, and then second fixation was done in 10% neutral buffered formalin solution for 1 h. The fixed tissues were dehydrated in a graded ethanol series and toluene, and then embedded in paraffin by using an automated tissue processor (Spin Tissue Processor STP-120, Thermo Scientific, Germany). .. Sections of 5 µm thickness were cut using a microtome (HM 430; Thermo Fisher Scientific, Germany), were stained with H & E. Slides were photographed under Olympus BX61 digital microscope (Olympus Co., Japan) attached with a computerized digital camera (DP72; Olympus, Tokyo, Japan) at various magnifications.

    Staining:

    Article Title: The developmental origin of heart size and shape differences in Astyanax mexicanus populations
    Article Snippet: 2.4 Paraffin embedding and sectioning To visualise the transparent embryos in paraffin, the embryos were transferred from 100% methanol to 0.1% eosin staining in 100% ethanol to stain the embryos for 30 s, followed by 3 washes with 100% ethanol. .. 7–12 µm sections were cut using a microtome (AO Spencer 820) and mounted onto Superfrost slides (Thermo Scientific), then dried overnight at 37 °C.

    Article Title: Gliclazide alone or in combination with atorvastatin ameliorated reproductive damage in streptozotocin-induced type 2 diabetic male rats
    Article Snippet: .. Sections of 5 µm thickness were cut using a microtome (HM 430; Thermo Fisher Scientific, Germany), were stained with H & E. Slides were photographed under Olympus BX61 digital microscope (Olympus Co., Japan) attached with a computerized digital camera (DP72; Olympus, Tokyo, Japan) at various magnifications. .. Paraffin embedded testis sections were immunostained for evaluation of intracellular junctions through determining expression and localization of claudin11 and occludin in seminiferous epithelium.

    Microscopy:

    Article Title: Gliclazide alone or in combination with atorvastatin ameliorated reproductive damage in streptozotocin-induced type 2 diabetic male rats
    Article Snippet: .. Sections of 5 µm thickness were cut using a microtome (HM 430; Thermo Fisher Scientific, Germany), were stained with H & E. Slides were photographed under Olympus BX61 digital microscope (Olympus Co., Japan) attached with a computerized digital camera (DP72; Olympus, Tokyo, Japan) at various magnifications. .. Paraffin embedded testis sections were immunostained for evaluation of intracellular junctions through determining expression and localization of claudin11 and occludin in seminiferous epithelium.

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    Thermo Fisher human liver microsome incubations mt 45
    Overview of the metabolic profiling of <t>MT-45</t> using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH), mouse urine (MU) from in vivo testing and authenticated human urine (HU) samples. Product ion spectra and structural elucidation data are provided in the supplementary material, section F
    Human Liver Microsome Incubations Mt 45, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher microsomal ho 1 blots
    Effect of treatment pre-H/R. LRS, MP4CO, MP4OX, or Hb (8 mL/kg) was infused into NY1DD sickle mice with implanted DSFCs 24 hours before H/R. Twenty-four hours after infusion, flowing venules in the subcutaneous skin were selected and mapped. Mice were then exposed to 1 hour of hypoxia (7% O 2 /93% N 2 ) and returned to room air. After 1 hour of reoxygenation, the same mapped venules were re-examined for blood flow, and the percentage of static (no flow) venules was calculated. Hemin pretreatment (40 µmols/day IP × 3 days) is a positive control known to prevent stasis by induction of <t>HO-1.</t> Values are means + SD. N = 4 mice per treatment except hemin (n = 6). * P
    Microsomal Ho 1 Blots, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher mouse hybridoma microsomes
    Overview of paired antibody HC-LC amplification using microsomes in water-in-oil emulsion droplets. a Antibody-expressing cell populations were used for microsome preparation. b Cells were lysed using a sucrose buffer with 5% digitonin and microsomes with rER-associated mRNAs were enriched using differential centrifugation. c Transmission electron microscopy showed enriched rER microsomes with multilamellar and unilamellar structures. The image was acquired from HEK 293T microsomes used for establishment of the method. Scale bar represents 100 nm. d HC and LC mRNAs were assembled by overlap extension RT-PCR to generate natively paired HC-LC amplicons using constant region primers for reverse transcription and variable region primers for overlap extension assembly. The location and orientation of the paired-end MiSeq reads on the amplicons are indicated by red arrows . e The assembly reaction was carried out within individual emulsion droplets with microsomes from single cells for clonal assembly of rER-associated mRNAs. f Nested PCR amplification with <t>hybridoma-specific</t> nested primers on the assembled DNA demonstrated strong enrichment of native HC-LC pairs when using emulsion PCR during the assembly reaction ( upper panel ), while a control showed random pairing of heavy and light chains when using conventional open PCR during the assembly reaction
    Mouse Hybridoma Microsomes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher surf4 enriched microsomes
    <t>Surf4</t> is essential for keeping AMELX, DSPP, and GH at low concentrations within ER. (A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells ( Surf4 KO ). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4 KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4 KO . Introduction of HA-tag slightly increased the M r of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4 KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELX myc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4 KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4 KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4 KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4 KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4 KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4 KO cells. Top panel: Surf4 KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca 2+ and pelleted at > 100,000 x g. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in > 100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4 KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4 KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P
    Surf4 Enriched Microsomes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of the metabolic profiling of MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH), mouse urine (MU) from in vivo testing and authenticated human urine (HU) samples. Product ion spectra and structural elucidation data are provided in the supplementary material, section F

    Journal: Forensic Toxicology

    Article Title: Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

    doi: 10.1007/s11419-018-0413-1

    Figure Lengend Snippet: Overview of the metabolic profiling of MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH), mouse urine (MU) from in vivo testing and authenticated human urine (HU) samples. Product ion spectra and structural elucidation data are provided in the supplementary material, section F

    Article Snippet: Pooled human liver microsome incubations MT-45, 2F-MT-45 and positive controls were incubated in pooled human liver microsome (pHLM; Thermo Fisher Scientific, Waltham, MA, USA) incubations at 37 °C with and without uridine 5′-diphosphoglucuronic acid (UDPGA).

    Techniques: In Vivo

    Chromatographic profiles and time-course data following incubation of MT-45 with a human hepatocytes after 120 min, and b human microsomes after 60 min obtained by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Metabolite labelling information can be found in Fig. 5 and Table 2

    Journal: Forensic Toxicology

    Article Title: Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

    doi: 10.1007/s11419-018-0413-1

    Figure Lengend Snippet: Chromatographic profiles and time-course data following incubation of MT-45 with a human hepatocytes after 120 min, and b human microsomes after 60 min obtained by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Metabolite labelling information can be found in Fig. 5 and Table 2

    Article Snippet: Pooled human liver microsome incubations MT-45, 2F-MT-45 and positive controls were incubated in pooled human liver microsome (pHLM; Thermo Fisher Scientific, Waltham, MA, USA) incubations at 37 °C with and without uridine 5′-diphosphoglucuronic acid (UDPGA).

    Techniques: Incubation, Liquid Chromatography, Mass Spectrometry

    Overview of the metabolic profiling of 2F-MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH) and mouse urine (MU) from in vivo testing. Product ion data and structural elucidation data are provided in supplementary material, section G

    Journal: Forensic Toxicology

    Article Title: Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

    doi: 10.1007/s11419-018-0413-1

    Figure Lengend Snippet: Overview of the metabolic profiling of 2F-MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH) and mouse urine (MU) from in vivo testing. Product ion data and structural elucidation data are provided in supplementary material, section G

    Article Snippet: Pooled human liver microsome incubations MT-45, 2F-MT-45 and positive controls were incubated in pooled human liver microsome (pHLM; Thermo Fisher Scientific, Waltham, MA, USA) incubations at 37 °C with and without uridine 5′-diphosphoglucuronic acid (UDPGA).

    Techniques: In Vivo

    Chemical structures of a MT-45, including the structural notation used for nuclear magnetic resonance (NMR) spectroscopy data using the system reported by [ 16 ], b 2F-MT-45, c 3F-MT-45, d 4F-MT-45, e 2FPPP (fluorolintane), f diphenidine and g 2-methoxphenidine (2-MXP)

    Journal: Forensic Toxicology

    Article Title: Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

    doi: 10.1007/s11419-018-0413-1

    Figure Lengend Snippet: Chemical structures of a MT-45, including the structural notation used for nuclear magnetic resonance (NMR) spectroscopy data using the system reported by [ 16 ], b 2F-MT-45, c 3F-MT-45, d 4F-MT-45, e 2FPPP (fluorolintane), f diphenidine and g 2-methoxphenidine (2-MXP)

    Article Snippet: Pooled human liver microsome incubations MT-45, 2F-MT-45 and positive controls were incubated in pooled human liver microsome (pHLM; Thermo Fisher Scientific, Waltham, MA, USA) incubations at 37 °C with and without uridine 5′-diphosphoglucuronic acid (UDPGA).

    Techniques: Nuclear Magnetic Resonance, Spectroscopy

    a 1 H NMR data for the seized sample, and b 1 H NMR data for the 2F-MT-45 reference standard. *Signals presumed to be originating from unknown excipient(s) present in the seized sample

    Journal: Forensic Toxicology

    Article Title: Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

    doi: 10.1007/s11419-018-0413-1

    Figure Lengend Snippet: a 1 H NMR data for the seized sample, and b 1 H NMR data for the 2F-MT-45 reference standard. *Signals presumed to be originating from unknown excipient(s) present in the seized sample

    Article Snippet: Pooled human liver microsome incubations MT-45, 2F-MT-45 and positive controls were incubated in pooled human liver microsome (pHLM; Thermo Fisher Scientific, Waltham, MA, USA) incubations at 37 °C with and without uridine 5′-diphosphoglucuronic acid (UDPGA).

    Techniques: Nuclear Magnetic Resonance

    Effect of treatment pre-H/R. LRS, MP4CO, MP4OX, or Hb (8 mL/kg) was infused into NY1DD sickle mice with implanted DSFCs 24 hours before H/R. Twenty-four hours after infusion, flowing venules in the subcutaneous skin were selected and mapped. Mice were then exposed to 1 hour of hypoxia (7% O 2 /93% N 2 ) and returned to room air. After 1 hour of reoxygenation, the same mapped venules were re-examined for blood flow, and the percentage of static (no flow) venules was calculated. Hemin pretreatment (40 µmols/day IP × 3 days) is a positive control known to prevent stasis by induction of HO-1. Values are means + SD. N = 4 mice per treatment except hemin (n = 6). * P

    Journal: Blood

    Article Title: MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice

    doi: 10.1182/blood-2013-02-486282

    Figure Lengend Snippet: Effect of treatment pre-H/R. LRS, MP4CO, MP4OX, or Hb (8 mL/kg) was infused into NY1DD sickle mice with implanted DSFCs 24 hours before H/R. Twenty-four hours after infusion, flowing venules in the subcutaneous skin were selected and mapped. Mice were then exposed to 1 hour of hypoxia (7% O 2 /93% N 2 ) and returned to room air. After 1 hour of reoxygenation, the same mapped venules were re-examined for blood flow, and the percentage of static (no flow) venules was calculated. Hemin pretreatment (40 µmols/day IP × 3 days) is a positive control known to prevent stasis by induction of HO-1. Values are means + SD. N = 4 mice per treatment except hemin (n = 6). * P

    Article Snippet: Microsomal HO-1 blots were stripped (Restore Stripping Buffer; Thermo Scientific, Waltham, MA) and reprobed with rabbit anti-GAPDH (Sigma-Aldrich).

    Techniques: Mouse Assay, Flow Cytometry, Positive Control

    Overview of paired antibody HC-LC amplification using microsomes in water-in-oil emulsion droplets. a Antibody-expressing cell populations were used for microsome preparation. b Cells were lysed using a sucrose buffer with 5% digitonin and microsomes with rER-associated mRNAs were enriched using differential centrifugation. c Transmission electron microscopy showed enriched rER microsomes with multilamellar and unilamellar structures. The image was acquired from HEK 293T microsomes used for establishment of the method. Scale bar represents 100 nm. d HC and LC mRNAs were assembled by overlap extension RT-PCR to generate natively paired HC-LC amplicons using constant region primers for reverse transcription and variable region primers for overlap extension assembly. The location and orientation of the paired-end MiSeq reads on the amplicons are indicated by red arrows . e The assembly reaction was carried out within individual emulsion droplets with microsomes from single cells for clonal assembly of rER-associated mRNAs. f Nested PCR amplification with hybridoma-specific nested primers on the assembled DNA demonstrated strong enrichment of native HC-LC pairs when using emulsion PCR during the assembly reaction ( upper panel ), while a control showed random pairing of heavy and light chains when using conventional open PCR during the assembly reaction

    Journal: Genome Medicine

    Article Title: Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes

    doi: 10.1186/s13073-018-0542-5

    Figure Lengend Snippet: Overview of paired antibody HC-LC amplification using microsomes in water-in-oil emulsion droplets. a Antibody-expressing cell populations were used for microsome preparation. b Cells were lysed using a sucrose buffer with 5% digitonin and microsomes with rER-associated mRNAs were enriched using differential centrifugation. c Transmission electron microscopy showed enriched rER microsomes with multilamellar and unilamellar structures. The image was acquired from HEK 293T microsomes used for establishment of the method. Scale bar represents 100 nm. d HC and LC mRNAs were assembled by overlap extension RT-PCR to generate natively paired HC-LC amplicons using constant region primers for reverse transcription and variable region primers for overlap extension assembly. The location and orientation of the paired-end MiSeq reads on the amplicons are indicated by red arrows . e The assembly reaction was carried out within individual emulsion droplets with microsomes from single cells for clonal assembly of rER-associated mRNAs. f Nested PCR amplification with hybridoma-specific nested primers on the assembled DNA demonstrated strong enrichment of native HC-LC pairs when using emulsion PCR during the assembly reaction ( upper panel ), while a control showed random pairing of heavy and light chains when using conventional open PCR during the assembly reaction

    Article Snippet: Emulsion RT-PCR assembly using mouse hybridoma microsomes We diluted 16 μL of resuspended microsomes from mixed hybridomas 5E4, KT13, and KT22 in 184 μL RT-PCR master mix containing 1× Verso 1-Step RT-PCR master mix (Thermo Scientific), 1× Verso enzyme mix (Thermo Scientific), 0.5 μg/μL BSA, 100 μg/mL cycloheximide, and primers for reverse transcription and HC and LC assembly (0.8 μM each of primers TitA_MID1_IgM_rev and TitB_MID12_IgK_rev; 0.16 μM each of primers OE_MHV_fwd and OE_MKV_fwd).

    Techniques: Amplification, Expressing, Centrifugation, Transmission Assay, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction, Nested PCR, Polymerase Chain Reaction

    Surf4 is essential for keeping AMELX, DSPP, and GH at low concentrations within ER. (A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells ( Surf4 KO ). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4 KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4 KO . Introduction of HA-tag slightly increased the M r of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4 KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELX myc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4 KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4 KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4 KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4 KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4 KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4 KO cells. Top panel: Surf4 KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca 2+ and pelleted at > 100,000 x g. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in > 100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4 KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4 KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Surf4 is essential for keeping AMELX, DSPP, and GH at low concentrations within ER. (A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells ( Surf4 KO ). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4 KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4 KO . Introduction of HA-tag slightly increased the M r of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4 KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELX myc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4 KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4 KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4 KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4 KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4 KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4 KO cells. Top panel: Surf4 KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca 2+ and pelleted at > 100,000 x g. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in > 100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4 KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4 KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: CRISPR, Plasmid Preparation, Affinity Purification, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Western Blot

    Model illustrating interaction of ER-ESCAPE motifs with high, modest, and no affinity for Surf4/Erv29p. (A) A green ball amino acid in spherical pocket denotes highest contribution of that residue to binding affinity such as a proline in number 2 position or a hydrophobic residue in position 1 (amino-terminus) or 3. Green half-ball plus red pyramid represents lower affinity interaction for that amino acid (e.g., serine in position 2), while the red cube denotes a negative contribution to binding affinity (e.g., acidic amino acid in position 1). High-affinity cargo present high-affinity contributions in all three positions, while modest- to low-affinity tripeptides have at least one mismatch. Nonbinding proteins such as chaperones or fibrillar collagens have two or three completely mismatching amino acids. (B) (1) High-affinity cargo (e.g., IPV) are bound to Surf4/Erv29p and exit ER before they accumulate to aggregate-forming concentrations. (2) Cargo with more modest ER-ESCAPE motifs (e.g., FSM) do not significantly bind to cargo receptor until (3) they accumulate to levels ≥ their binding constant. Only at that point do they remain bound long enough to remain in COPII vesicle at levels significantly greater than bulk flow. (4) illustrates cargo starting with nonbinding amino-terminal tripeptides (e.g., QEE) cannot exit ER more efficiently than their concentration in ER lumenal fluid in equilibrium with the small amount of exit vesicle fluid (bulk flow). (5) Fibrillar collagens are too large for standard COPII exit vesicles and must use more voluminous TANGO1/cTAGE5-associated exit vesicles. Large fibrillar collagens often start with nonbinding motif (e.g., QEE) to keep them from binding Surf4 and partially entering smaller COPII vesicles. COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; IPV, isoleucine-proline-valine; QEE, glutamine–glutamic acid–glutamic acid; Surf4, surfeit locus protein 1; TANGO1, transport and Golgi organization 1.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Model illustrating interaction of ER-ESCAPE motifs with high, modest, and no affinity for Surf4/Erv29p. (A) A green ball amino acid in spherical pocket denotes highest contribution of that residue to binding affinity such as a proline in number 2 position or a hydrophobic residue in position 1 (amino-terminus) or 3. Green half-ball plus red pyramid represents lower affinity interaction for that amino acid (e.g., serine in position 2), while the red cube denotes a negative contribution to binding affinity (e.g., acidic amino acid in position 1). High-affinity cargo present high-affinity contributions in all three positions, while modest- to low-affinity tripeptides have at least one mismatch. Nonbinding proteins such as chaperones or fibrillar collagens have two or three completely mismatching amino acids. (B) (1) High-affinity cargo (e.g., IPV) are bound to Surf4/Erv29p and exit ER before they accumulate to aggregate-forming concentrations. (2) Cargo with more modest ER-ESCAPE motifs (e.g., FSM) do not significantly bind to cargo receptor until (3) they accumulate to levels ≥ their binding constant. Only at that point do they remain bound long enough to remain in COPII vesicle at levels significantly greater than bulk flow. (4) illustrates cargo starting with nonbinding amino-terminal tripeptides (e.g., QEE) cannot exit ER more efficiently than their concentration in ER lumenal fluid in equilibrium with the small amount of exit vesicle fluid (bulk flow). (5) Fibrillar collagens are too large for standard COPII exit vesicles and must use more voluminous TANGO1/cTAGE5-associated exit vesicles. Large fibrillar collagens often start with nonbinding motif (e.g., QEE) to keep them from binding Surf4 and partially entering smaller COPII vesicles. COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; IPV, isoleucine-proline-valine; QEE, glutamine–glutamic acid–glutamic acid; Surf4, surfeit locus protein 1; TANGO1, transport and Golgi organization 1.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Binding Assay, Flow Cytometry, Concentration Assay

    When not in functional excess, Surf4 prioritizes ER exit of cargo with stronger ER-ESCAPE motif. (A) Western blot analyses of cell extracts show steady-state levels of GH starting with strong ER-ESCAPE motif: APV

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: When not in functional excess, Surf4 prioritizes ER exit of cargo with stronger ER-ESCAPE motif. (A) Western blot analyses of cell extracts show steady-state levels of GH starting with strong ER-ESCAPE motif: APV

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Functional Assay, Western Blot

    Immunofluorescence microscopy of HEK293A cells shows Surf4 accumulates in and around ERESs. (A) Fluorescent signal for endogenous Surf4 (green) was strongest at punctate structures positive for ERES marker Sec23 (red). Note additional Surf4 fluorescence in weblike structures surrounding ERES. (B) HA-Surf4 signal (green) was observed within the ERGIC (ERGIC-53, red). (C) HA-Surf4 (green) showed only low levels of colocalization with cis -Golgi marker, giantin (red). (D) Mutation of proposed COPI recycling motif by replacement of two of three near-carboxy-terminal lysines to alanines (HA-Surf4-AAK, green) increased colocalization with cis -Golgi marker, giantin (red). (E) Newly synthesized HA-Surf4 (green) was found at low levels in the rER (Sec61 marker, red). (F) Surf4 (green) did not colocalize with chaperone, calnexin (red), in the quality control domain. HEK293A cells were transfected with wild-type HA-tagged Surf4 plasmid (B, C, E), or Surf4 KO HEK293A cells were transfected with carboxyl-terminal di-lysine mutation, HA- Surf4 -AAK (D), 18 hr prior to fixation. Bars = 5μm. The cells in each panel are shown 3 times, first with organelle marker, then Surf4 and final panel (with magnified insert) showing overlap. Alexa Fluor secondary antibodies were used for detection. Images were obtained using an LSM 780 (Carl Zeiss) confocal microscope (488 and 561 nm excitation lines; 500–560 and 600–660 nm capture) and Zeiss Axio Imager Z1 with Apotome 2 (single Z stack slice). Images were analyzed using Zeiss Zen software. AAK, alanine-alanine-lysine; COP, coat protein complex I; ERES, endoplasmic reticulum exit site; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293A; rER, rough ER; Surf4, surfeit locus protein 4.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Immunofluorescence microscopy of HEK293A cells shows Surf4 accumulates in and around ERESs. (A) Fluorescent signal for endogenous Surf4 (green) was strongest at punctate structures positive for ERES marker Sec23 (red). Note additional Surf4 fluorescence in weblike structures surrounding ERES. (B) HA-Surf4 signal (green) was observed within the ERGIC (ERGIC-53, red). (C) HA-Surf4 (green) showed only low levels of colocalization with cis -Golgi marker, giantin (red). (D) Mutation of proposed COPI recycling motif by replacement of two of three near-carboxy-terminal lysines to alanines (HA-Surf4-AAK, green) increased colocalization with cis -Golgi marker, giantin (red). (E) Newly synthesized HA-Surf4 (green) was found at low levels in the rER (Sec61 marker, red). (F) Surf4 (green) did not colocalize with chaperone, calnexin (red), in the quality control domain. HEK293A cells were transfected with wild-type HA-tagged Surf4 plasmid (B, C, E), or Surf4 KO HEK293A cells were transfected with carboxyl-terminal di-lysine mutation, HA- Surf4 -AAK (D), 18 hr prior to fixation. Bars = 5μm. The cells in each panel are shown 3 times, first with organelle marker, then Surf4 and final panel (with magnified insert) showing overlap. Alexa Fluor secondary antibodies were used for detection. Images were obtained using an LSM 780 (Carl Zeiss) confocal microscope (488 and 561 nm excitation lines; 500–560 and 600–660 nm capture) and Zeiss Axio Imager Z1 with Apotome 2 (single Z stack slice). Images were analyzed using Zeiss Zen software. AAK, alanine-alanine-lysine; COP, coat protein complex I; ERES, endoplasmic reticulum exit site; ERGIC, endoplasmic reticulum–Golgi intermediate compartment; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293A; rER, rough ER; Surf4, surfeit locus protein 4.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Immunofluorescence, Microscopy, Marker, Fluorescence, Mutagenesis, Synthesized, Transfection, Plasmid Preparation, Software

    Exit of highest affinity cargo is prioritized in ERES lacking an excess of Surf4/Erv29p. Cargo receptors must have a high-affinity conformation to bind cargo in the ER and a low-affinity conformation to release cargo in fully formed exit vesicle or upon fusing with ERGIC/Golgi. High-affinity panel (A) illustrates a model whereby cargo receptors in the vicinity of ERESs have the ability to bind cargo before physically entering COPII vesicle, while the low-affinity panel (B) represents an alternative model in which the receptor is in its low-affinity state until interacting with elements of COPII vesicle (e.g., Sec24). In both cases, when there is an excess of cargo for local population of receptors, high-affinity cargo occupies available receptors, while lower-affinity cargo continues to build in concentration. This aids in keeping the most problematical proteins below their aggregation concentrations. Similarly, modest-affinity cargo, when their concentration becomes ≥ binding constant, occupy any available receptors before cargo with still-lower-affinity ER-ESCAPE motifs. This process delays aggregate formation until cargo receptors can be brought into balance with local/total cargo loading. Nonbinding cargo continue to exit solely by diffusion/equilibrium between fluids of COPII vesicle and ERES lumen (bulk flow). COPII, coat protein complex II; ER, endoplasmic reticulum; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERGIC, ER-Golgi intermediate compartment; Erv29p, ER-derived vesicles protein 29; Surf4, surfeit locus protein 4.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Exit of highest affinity cargo is prioritized in ERES lacking an excess of Surf4/Erv29p. Cargo receptors must have a high-affinity conformation to bind cargo in the ER and a low-affinity conformation to release cargo in fully formed exit vesicle or upon fusing with ERGIC/Golgi. High-affinity panel (A) illustrates a model whereby cargo receptors in the vicinity of ERESs have the ability to bind cargo before physically entering COPII vesicle, while the low-affinity panel (B) represents an alternative model in which the receptor is in its low-affinity state until interacting with elements of COPII vesicle (e.g., Sec24). In both cases, when there is an excess of cargo for local population of receptors, high-affinity cargo occupies available receptors, while lower-affinity cargo continues to build in concentration. This aids in keeping the most problematical proteins below their aggregation concentrations. Similarly, modest-affinity cargo, when their concentration becomes ≥ binding constant, occupy any available receptors before cargo with still-lower-affinity ER-ESCAPE motifs. This process delays aggregate formation until cargo receptors can be brought into balance with local/total cargo loading. Nonbinding cargo continue to exit solely by diffusion/equilibrium between fluids of COPII vesicle and ERES lumen (bulk flow). COPII, coat protein complex II; ER, endoplasmic reticulum; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERGIC, ER-Golgi intermediate compartment; Erv29p, ER-derived vesicles protein 29; Surf4, surfeit locus protein 4.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Concentration Assay, Binding Assay, Diffusion-based Assay, Flow Cytometry, Derivative Assay

    Relative affinities of ER-ESCAPE motifs for cargo receptor by analyses of GH steady-state levels. (A) Pre-confluent cells were separately transfected with expression plasmids encoding human GH with 60 different ER-ESCAPE motifs as noted. Eighteen hr posttransfection, cells were washed, extracted with lysis buffer, and analyzed by GH ELISA. GH values (ng GH/mg protein) were normalized within each experiment to the amount of IPV-GH. Each histogram bar represents mean ± SEM. of at least three independent transfections for each construct. (Means of triplicate GH ELISA analyses were used for each extract). Larger error bars among poorest ER-ESCAPE motifs may reflect a variable amount of aggregate formation by larger amounts of accumulated GH. Note that Φ-P-Φ ER-ESCAPE motifs are among the most efficient at trafficking GH, while those including acidic amino acids or glutamines are much less effective. Substitution by positively charged amino acids (generally, R better than K) retained effective trafficking, while loss of proline in position 2 was otherwise detrimental. (B) Prediction of value that each amino acid in positions 1, 2, and 3 adds to the quality of the ER-ESCAPE motif binding to Surf4 based on combination of ELISA and database search results. Note that some combinations may give results not predicted by simply summing a tripeptide’s three individual amino acids contributions indicated by this panel. Φ-P-Φ, hydrophobic-proline-hydrophobic; APV, alanine-proline-valine; DYP, aspartic acid-tyrosine-proline; EEE, glutamic acid–glutamic acid–glutamic acid; EEI, glutamic acid–glutamic acid–isoleucine; EET, glutamic acid–glutamic acid–threonine; EGT, glutamic acid-glycine-threonine; EPA, glutamic acid-proline-alanine; EPT, glutamic acid-proline-threonine; EPV, glutamic acid-proline-valine; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; EST, glutamic acid-serine-threonine; FGT, phenylalanine-glycine-threonine; FLT, phenylalanine-leucine-threonine; FPE, phenylalanine-proline-glutamic acid; FPR, phenylalanine-proline-arginine; FPT, phenylalanine-proline-threonine; FPV, phenylalanine-proline-valine; FQV, phenylalanine-glutamine-valine; FSM, phenylalanine-serine-methionine; FST, phenylalanine-threonine-valine; FTV, phenylalanine-threonine-valine; FVN, phenylalanine-valine-asparagine; GH, growth hormone; GPV, glycine-proline-valine; HSV, histidine-serine-valine; IEV, isoleucine-glycine-valine; IGV, isoleucine-glycine-valine; ILV, isoleucine-leucine-valine; INV, isoleucine-asparagine-valine; IPA, isoleucine-proline-alanine; IPD, isoleucine-proline-aspartic acid; IPE, isoleucine-proline-glutamic acid; IPP, isoleucine-proline-proline; IPS, isoleucine-proline-serine; IPV, isoleucine-proline-valine; IRV, isoleucine-arginine-valine; ISH, isoleucine-serine-histidine; ISP, isoleucine-serine-proline; ISQ, isoleucine-serine-glutamine; ISR, isoleucine-serine-arginine; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; KAV, lysine-alanine-valine; KGV, lysine-glycine-valine; KSV, lysine-serine-valine; KVH, lysine-valine-histidine; NPV, asparagine-proline-valine; QPV, glutamine-proline-valine; QQV, glutamine-glutamine-valine; QSV, glutamine-serine-valine; RGV, arginine-glycine-valine; RLV, arginine-leucine-valine; RPK, arginine-proline-lysine; RPV, arginine-proline-valine; RRR, arginine-arginine-arginine; RSV, arginine-serine-valine; SLT, serine-leucine-threonine; SPT, serine-proline-threonine; SPV, serine-proline-valine; SRT, serine-arginine-threonine; SST, serine-serine-threonine; Surf4, surfeit locus protein 4; YPY, tyrosine-proline-tyrosine.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Relative affinities of ER-ESCAPE motifs for cargo receptor by analyses of GH steady-state levels. (A) Pre-confluent cells were separately transfected with expression plasmids encoding human GH with 60 different ER-ESCAPE motifs as noted. Eighteen hr posttransfection, cells were washed, extracted with lysis buffer, and analyzed by GH ELISA. GH values (ng GH/mg protein) were normalized within each experiment to the amount of IPV-GH. Each histogram bar represents mean ± SEM. of at least three independent transfections for each construct. (Means of triplicate GH ELISA analyses were used for each extract). Larger error bars among poorest ER-ESCAPE motifs may reflect a variable amount of aggregate formation by larger amounts of accumulated GH. Note that Φ-P-Φ ER-ESCAPE motifs are among the most efficient at trafficking GH, while those including acidic amino acids or glutamines are much less effective. Substitution by positively charged amino acids (generally, R better than K) retained effective trafficking, while loss of proline in position 2 was otherwise detrimental. (B) Prediction of value that each amino acid in positions 1, 2, and 3 adds to the quality of the ER-ESCAPE motif binding to Surf4 based on combination of ELISA and database search results. Note that some combinations may give results not predicted by simply summing a tripeptide’s three individual amino acids contributions indicated by this panel. Φ-P-Φ, hydrophobic-proline-hydrophobic; APV, alanine-proline-valine; DYP, aspartic acid-tyrosine-proline; EEE, glutamic acid–glutamic acid–glutamic acid; EEI, glutamic acid–glutamic acid–isoleucine; EET, glutamic acid–glutamic acid–threonine; EGT, glutamic acid-glycine-threonine; EPA, glutamic acid-proline-alanine; EPT, glutamic acid-proline-threonine; EPV, glutamic acid-proline-valine; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; EST, glutamic acid-serine-threonine; FGT, phenylalanine-glycine-threonine; FLT, phenylalanine-leucine-threonine; FPE, phenylalanine-proline-glutamic acid; FPR, phenylalanine-proline-arginine; FPT, phenylalanine-proline-threonine; FPV, phenylalanine-proline-valine; FQV, phenylalanine-glutamine-valine; FSM, phenylalanine-serine-methionine; FST, phenylalanine-threonine-valine; FTV, phenylalanine-threonine-valine; FVN, phenylalanine-valine-asparagine; GH, growth hormone; GPV, glycine-proline-valine; HSV, histidine-serine-valine; IEV, isoleucine-glycine-valine; IGV, isoleucine-glycine-valine; ILV, isoleucine-leucine-valine; INV, isoleucine-asparagine-valine; IPA, isoleucine-proline-alanine; IPD, isoleucine-proline-aspartic acid; IPE, isoleucine-proline-glutamic acid; IPP, isoleucine-proline-proline; IPS, isoleucine-proline-serine; IPV, isoleucine-proline-valine; IRV, isoleucine-arginine-valine; ISH, isoleucine-serine-histidine; ISP, isoleucine-serine-proline; ISQ, isoleucine-serine-glutamine; ISR, isoleucine-serine-arginine; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; KAV, lysine-alanine-valine; KGV, lysine-glycine-valine; KSV, lysine-serine-valine; KVH, lysine-valine-histidine; NPV, asparagine-proline-valine; QPV, glutamine-proline-valine; QQV, glutamine-glutamine-valine; QSV, glutamine-serine-valine; RGV, arginine-glycine-valine; RLV, arginine-leucine-valine; RPK, arginine-proline-lysine; RPV, arginine-proline-valine; RRR, arginine-arginine-arginine; RSV, arginine-serine-valine; SLT, serine-leucine-threonine; SPT, serine-proline-threonine; SPV, serine-proline-valine; SRT, serine-arginine-threonine; SST, serine-serine-threonine; Surf4, surfeit locus protein 4; YPY, tyrosine-proline-tyrosine.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Transfection, Expressing, Lysis, Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Indirect Immunoperoxidase Assay, In Situ Hybridization, Single-particle Tracking

    Examples of starting tripeptides in soluble proteins that are predicted not to interact with Surf4/Erv29p. for accession number, species name, and brief representative sequence.) Color-coding based on relative contribution of each amino acid position to strength of the ER-ESCAPE motif. CALR, calreticulin; COL1A1, collagen type 1 alpha 1; COL2A1, collagen type 2 alpha 1; COL3A1, collagen type 3 alpha 1; COL6A1, collagen type 6 alpha 1; COL1A2, collagen type 6 alpha 2; COL6A3, collagen type 6 alpha 3; COL7A1, collagen type 7 alpha 1; COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERO1, ER oxidoreductase 1; Erv29p, ER-derived vesicles protein 29; F-GRP78, fungal glucose-regulated protein 78; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94; NCBI, National Center for Biotechnology Information; PDI, protein disulfide isomerase; PDIA2, PDI family A member 2; PDIA4, PDI family A member 4; Surf4, surfeit locus protein 4; TANGO 1, transport and Golgi organization 1.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: Examples of starting tripeptides in soluble proteins that are predicted not to interact with Surf4/Erv29p. for accession number, species name, and brief representative sequence.) Color-coding based on relative contribution of each amino acid position to strength of the ER-ESCAPE motif. CALR, calreticulin; COL1A1, collagen type 1 alpha 1; COL2A1, collagen type 2 alpha 1; COL3A1, collagen type 3 alpha 1; COL6A1, collagen type 6 alpha 1; COL1A2, collagen type 6 alpha 2; COL6A3, collagen type 6 alpha 3; COL7A1, collagen type 7 alpha 1; COPII, coat protein complex II; ER, endoplasmic reticulum; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; ERO1, ER oxidoreductase 1; Erv29p, ER-derived vesicles protein 29; F-GRP78, fungal glucose-regulated protein 78; GRP78, glucose-regulated protein 78; GRP94, glucose-regulated protein 94; NCBI, National Center for Biotechnology Information; PDI, protein disulfide isomerase; PDIA2, PDI family A member 2; PDIA4, PDI family A member 4; Surf4, surfeit locus protein 4; TANGO 1, transport and Golgi organization 1.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Sequencing, Derivative Assay

    APV-GH interacts with digitonin-permeabilized Surf4 microsomes with half-maximal binding of 200–300 nM. (A) Microsomes made from Surf4 KO cells with (Lanes 1–4) or without (Lanes 5–8) expression of Surf4 expression by transfection for 24 hr. Half of the microsome aliquots (Lanes 3, 4, 7, and 8) were permeabilized with CEB (digitonin) for 30 min. As indicted, aliquots were incubated for 1 hr with 400 nM APV-GH or 400 nM EET-GH and briefly washed. Only combination of Surf4-expressing microsomes + digitonin + APV-GH resulted in significant increases ( > 5-fold) in detection by GH ELISA associated with the final > 100,000 x g microsome pellet. (B) Preparation of microsomes from HA-Surf4-transfected HEK293A cells were incubated with magnetic beads precoated with antibodies to the cytosolic, carboxy-terminal domain of Surf4. Equal aliquots of Surf4 microsome beads were titrated with indicated concentration of digitonin (or CEB) for 30 min before incubation for 1 hr with 400 nM APV-GH, brief wash, and processing for GH ELISA analyses. Dose-response results show that CEB and ≥ 30 μg/ml digitonin were effective at permeabilizing microsomes for binding of APV-GH. Insert: Western blot shows that bead-associated microsomes contained both HA-Surf4 and the ERES marker, Sec23. (C) Equal aliquots of CEB-treated, Surf4 microsome/beads were incubated with increasing concentrations of APV-GH or EET-GH. APV-GH showed saturable binding characteristics with half-maximal binding at around 200–300 nM. EET-GH showed background levels of binding. (D) Equal aliquots of Surf4 microsome/beads were permeabilized with CEB (except first lane), incubated for 1 hr with 400 nM GH starting with indicated tripeptides, briefly washed, and analyzed by GH ELISA. Highest level of binding was with strong ER-ESCAPE motif APV-GH, followed by three modest binding motifs, FSM-GH, ISV-GH, and ITV-GH. The two acidic motifs, EET-GH and EEE-GH, bound at low levels also observed for microsome/beads not permeabilized by detergent. Each histogram bar represents mean ± SEM of transfections with each construct ( n ≥ 7) with statistical comparisons to APV-GH (** p ≤ 0.001, * p ≤0.01) or to EET-GH (°° p ≤ 0.001). APV, alanine-proline-valine; CEB, Cytosol Extraction Buffer; EEE, glutamic acid–glutamic acid–glutamic acid; EET, glutamic acid–glutamic acid–threonine; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; GH, growth hormone; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; Surf4, surfeit locus protein 4.

    Journal: PLoS Biology

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    doi: 10.1371/journal.pbio.2005140

    Figure Lengend Snippet: APV-GH interacts with digitonin-permeabilized Surf4 microsomes with half-maximal binding of 200–300 nM. (A) Microsomes made from Surf4 KO cells with (Lanes 1–4) or without (Lanes 5–8) expression of Surf4 expression by transfection for 24 hr. Half of the microsome aliquots (Lanes 3, 4, 7, and 8) were permeabilized with CEB (digitonin) for 30 min. As indicted, aliquots were incubated for 1 hr with 400 nM APV-GH or 400 nM EET-GH and briefly washed. Only combination of Surf4-expressing microsomes + digitonin + APV-GH resulted in significant increases ( > 5-fold) in detection by GH ELISA associated with the final > 100,000 x g microsome pellet. (B) Preparation of microsomes from HA-Surf4-transfected HEK293A cells were incubated with magnetic beads precoated with antibodies to the cytosolic, carboxy-terminal domain of Surf4. Equal aliquots of Surf4 microsome beads were titrated with indicated concentration of digitonin (or CEB) for 30 min before incubation for 1 hr with 400 nM APV-GH, brief wash, and processing for GH ELISA analyses. Dose-response results show that CEB and ≥ 30 μg/ml digitonin were effective at permeabilizing microsomes for binding of APV-GH. Insert: Western blot shows that bead-associated microsomes contained both HA-Surf4 and the ERES marker, Sec23. (C) Equal aliquots of CEB-treated, Surf4 microsome/beads were incubated with increasing concentrations of APV-GH or EET-GH. APV-GH showed saturable binding characteristics with half-maximal binding at around 200–300 nM. EET-GH showed background levels of binding. (D) Equal aliquots of Surf4 microsome/beads were permeabilized with CEB (except first lane), incubated for 1 hr with 400 nM GH starting with indicated tripeptides, briefly washed, and analyzed by GH ELISA. Highest level of binding was with strong ER-ESCAPE motif APV-GH, followed by three modest binding motifs, FSM-GH, ISV-GH, and ITV-GH. The two acidic motifs, EET-GH and EEE-GH, bound at low levels also observed for microsome/beads not permeabilized by detergent. Each histogram bar represents mean ± SEM of transfections with each construct ( n ≥ 7) with statistical comparisons to APV-GH (** p ≤ 0.001, * p ≤0.01) or to EET-GH (°° p ≤ 0.001). APV, alanine-proline-valine; CEB, Cytosol Extraction Buffer; EEE, glutamic acid–glutamic acid–glutamic acid; EET, glutamic acid–glutamic acid–threonine; ERES, ER exit site; ER-ESCAPE motif, ER-Exit by Soluble Cargo using Amino-terminal Peptide-Encoding motif; FSM, phenylalanine-serine-methionine; GH, growth hormone; HA, hemagglutinin; HEK293A, human embryonic kidney cell line 293; ISV, isoleucine-serine-valine; ITV, isoleucine-threonine-valine; Surf4, surfeit locus protein 4.

    Article Snippet: Surf4-enriched microsomes were prepared using 10 μg affinity-purified rabbit anti-Surf4-CT prebound to each 50 μl aliquot of magnetic Protein G-Dynabeads (Thermo Fisher Scientific).

    Techniques: Binding Assay, Expressing, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Magnetic Beads, Concentration Assay, Western Blot, Marker, Construct