Journal: PLOS Pathogens

Article Title: Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

doi: 10.1371/journal.ppat.1013867

Figure Lengend Snippet: (A) Western blot showing the amplification of PK-resistant PrP Sc from brain homogenate PMCA reactions supplemented with DMSO or NGI-1. (B) Representative flow cytometry plot showing effect of siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b combination on surface PrP C levels in WT CAD5 cells. (C) Quantification of flow cytometry PrP C surface expression in undifferentiated CAD5 cells treated with siRNAs targeted at Stt3a , Stt3b , or Stt3a and Stt3b in combination. Points represent individual samples from biological triplicates. Asterisks represent significance values from unpaired t-tests as follows: **p 0.01, ns = not significant. (D) Western blot showing the loss of PK-resistant PrP Sc in the lysates from CAD5 cells chronically infected with 22L prions after siRNA knockdown of Stt3a , Stt3b , or Stt3a and Stt3b in combination.

Article Snippet: Western blot was performed on cell lysate to assess PK-resistant PrP Sc as described above or to assess knockdown efficiency of STT3A or STT3B using STT3A monoclonal antibody 1E2B12 (Proteintech, Rosemont, IL, USA) or STT3B polyclonal antibody (Proteintech, Cat # 15323–1-AP) as primary antibodies and horseradish peroxidase-labeled sheep-anti-mouse secondary antibody (Cytiva) or goat-anti-rabbit secondary antibody (BioRad, Hercules, CA, USA, Cat # 1706515) at manufacturers’ recommended dilutions

Techniques: Western Blot, Amplification, Flow Cytometry, Knockdown, Expressing, Infection

Journal: Journal of cell science

Article Title: Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.

doi: 10.1242/jcs.094599

Figure Lengend Snippet: Fig. 6. KCP2 and STT3A are not readily extracted from the RAMP fraction. HeLa cells were solubilised in a buffer containing 1.5% digitonin and adjusted to 25, 75, 150 or 300 mM NaCl. After ultracentrifugation, soluble supernatant fractions and their corresponding RAMP fractions were isolated. The distribution of KCP2, STT3A, STT3B, OST48, Rib I, Sec61a and Rpl17 between these fractions was assessed by infrared fluorescent western blotting. Fluorescent signals were quantified and the amount of extracted protein was expressed as a percentage of total protein for each fraction.

Article Snippet: The plasmid encoding STT3A was purchased from OriGene Technologies.

Techniques: Isolation, Western Blot

Journal: Journal of cell science

Article Title: Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.

doi: 10.1242/jcs.094599

Figure Lengend Snippet: Fig. 7. KCP2 is assembled into distinct isoforms of the OST complex containing STT3A as a catalytic subunit. (A) Infrared western blot analysis of RIPA lysates (40 mg total protein per lane) of various cultured cell lines (HeLa, cervical epithelial carcinoma; HT1080, fibrosarcoma breast cancer; HEK293, human embryonic kidney; HepG2, hepatoma) with the anti-KCP2, anti-STT3A, anti-STT3B, anti-OST48, anti-Sec61a and anti-DAD1 antibodies. Alpha-tubulin was detected as a loading control. (B,C) Infrared western blotting after BN-PAGE analysis of digitonin-solubilised HepG2 cells with antibodies to Sec61a, KCP2, STT3A, STT3B, DAD1, OST48 and DC2 as labelled. The protein complexes containing STT3A (OSTC470 and OSTC500), STT3B (OSTC550) and Sec61a (Sec61C) are indicated at the right.

Article Snippet: The plasmid encoding STT3A was purchased from OriGene Technologies.

Techniques: Western Blot, Cell Culture, Control

Journal: Journal of cell science

Article Title: Keratinocyte-associated protein 2 is a bona fide subunit of the mammalian oligosaccharyltransferase.

doi: 10.1242/jcs.094599

Figure Lengend Snippet: Fig. 8. FLAG–KCP2 is associated specifically with core OST subunits. (A,B,D,E) HEK293 cells were (co-)transfected with expression plasmids for the proteins indicated, and digitonin lysates were immunoprecipitated with mouse anti-FLAG. One quarter of the total lysate used for each immunoprecipitation reaction (lanes 1–3) and the immunopurified proteins (lanes 4–6) were analysed by immunoblotting (IB) with the antibodies indicated, followed by re- immunoblotting for additional components (reIB). When detecting the endogenous OST48 signal (A,B,D) or the FLAG–gp78 signal (E), the earlier signals could still be seen in totals and in anti-FLAG precipitates (indicated in grey lettering). In A and D, a non-specific band always detected in total lysate samples by the anti-STT3A antibody is indicated by asterisks. This non-specific immunoreaction product migrates in the vicinity of OST48, which is marked by black circles for clarity. Cross-reacting bands occasionally detected by the anti-KCP2 or anti-FLAG antibodies are indicated by arrowheads. (C) The graph shows the relative amount of FLAG–KCP2-interacting STT3A (grey bars) and STT3B (black bars). The levels of STT3A and STT3B in the immunoprecipitates were expressed relative to the corresponding total levels, and then normalised to the levels of the recovered FLAG–KCP2 in the same samples.

Article Snippet: The plasmid encoding STT3A was purchased from OriGene Technologies.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Proteomic Analysis Identifies Membrane Proteins Dependent on the ER Membrane Protein Complex

doi: 10.1016/j.celrep.2019.08.006

Figure Lengend Snippet:

Article Snippet: STT3A pEGFP-N2 , Addgene , 62025.

Techniques: Recombinant, Modification, Protease Inhibitor, Membrane, BIA-KA, Software

Journal: mBio

Article Title: Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

doi: 10.1128/mBio.00939-17

Figure Lengend Snippet: The CRISPR-Cas9 screen reveals the oligosaccharyltransferase complex as essential to DENV propagation. (A) Depiction of the whole-genome CRISPR screen selecting for knockout cells resistant to dengue virus infection. (B) Diagram of the two OST complexes that contain either STT3A or STT3B. Subunits of the OST complex shown in blue were significantly enriched in the screen. (C) Table of the hits from the screen showing the gene identifier (ID), P value, and false-discovery rate as calculated by MAGeCK analysis.

Article Snippet: The cDNA clone for STT3A was purchased from OriGene (RC201991; Rockville, MD).

Techniques: CRISPR, Knock-Out, Virus, Infection

Journal: mBio

Article Title: Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

doi: 10.1128/mBio.00939-17

Figure Lengend Snippet: The catalytic activities of STT3A or STT3B are not required for DENV replication. (A) Huh7.5.1 cells were transduced with a lentivirus encoding a puromycin resistance marker, Cas9, and a single guide RNA (sgRNA) targeting the indicated genes. The sgRNAs targeting GFP act as a negative control. Two independent sgRNAs (indicated by 1 or 2 below the bars in the bar graph) were used per target gene to generate knockout cell pools. Cells were infected with luc-DENV for 3 days, and luciferase activity was measured in relative light units (RLU). Western blots for the indicated proteins are shown below the bar graph. Arrows indicate nonspecific bands. (B and C) Knockout cell pools were transduced with lentiviral vectors to rescue expression of the indicated protein. “STT3A dead” is a catalytically inactive W526A D527A double mutant. “STT3B dead” is a catalytically inactive W605A D606A double mutant with a C-terminal V5 tag. Transient transfection of constructs to express pSAP and SHBG were used to assess STT3A and STT3B catalytic activity. Western blots below the bar graph show the expression of the indicated proteins. wt and WT, wild type. (D) Knockout cell pools were transiently transfected with a luc-DENV replicon, and luciferase activity was measured at 4 h, 48 h, and 72 h posttransfection (hpt). Data are plotted as a ratio of RLU to the RLU at 4 hpt to control for transfection efficiency. GDD indicates a polymerase dead replicon with replacement of the GNN active site sequence in the NS5 RNA polymerase by GDD. (E) Cell viability for knockout cell pools was measured by Cell-Titer Glo over the course of 4 days for three independent wells. Data are normalized to day 1 values. The values for the parental Huh7.5.1 cells (P) are shown. In panels A to E, data are expressed as means plus standard deviations (SD) (error bars) for three independent biological replicates. In panels A to D, statistical significance was determined by Student’s t test and indicated by asterisks as follows: *, P < 0.05; **, P < 0.005; ***, P < 0.0005. In panel A, the means were compared to the mean for GFP sgRNA 2. In panels B and C, the means were compared to the means of their respective knockouts. In panel D, the means were compared to the mean for the GFP control.

Article Snippet: The cDNA clone for STT3A was purchased from OriGene (RC201991; Rockville, MD).

Techniques: Transduction, Marker, Negative Control, Knock-Out, Infection, Luciferase, Activity Assay, Western Blot, Expressing, Mutagenesis, Transfection, Construct, Control, Sequencing