human embryonic fibroblasts hek293  (Thermo Fisher)

Journal: Molecular cell

Article Title: Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis.

doi: 10.1016/j.molcel.2018.08.019

Figure Lengend Snippet: Figure 2. Mapping Polysome-Interacting Proteins (A) Top: overlap of proteins (excluding ribosomal proteins) identified in polysome fractions in our study and the so-called mammalian riboproteome. Bottom: complexes with cytosolic RPs (green bars, including ribosomal proteins) and several non-ribosomal complexes (purple bars) are significantly enriched. (B) To identify ribosome-associated proteins, profiles of individual proteins are compared with the polysome consensus profile by computing MSD values. (C) Observed distribution of MSD values in HEK293 cells (green). Cytosolic (red) and mitochondrial RPs (purple) can be easily separated. The MSD value dis- tribution of an exemplarily shuffled dataset is depicted in gray. Multiple shuffling operations were used to define cutoffs (nominal false discovery rate [FDR] = 0). The insets show the reproducibility of MSD values between replicates.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 3P for B cells This paper ProteomeXchange: PXD009267 pSILAC in B cells This paper ProteomeXchange: PXD009276 PRM assay in B cells This paper ProteomeXchange: PXD010029 RNA-seq and ribo-seq datasets This paper GEO: GSE112187 Human UniprotKB/Swiss-Prot data base (Human UniProt 2014-10) N/A https://www.uniprot.org/proteomes/ Mouse UniprotKB/Swiss-Prot data base (Mouse UniProt 2014-10) N/A https://www.uniprot.org/proteomes/ Riboproteome data Reschke et al., 2013 N/A Ribosome profiling data (mitosis versus S phase) Stumpf et al., 2013 N/A Protein complex annotation data (CORUM downloaded Jan/2017) Ruepp et al., 2010 https://mips.helmholtz-muenchen.de/corum/ STRING protein interaction database Szklarczyk et al., 2017 https://string-db.org/ RNAi data for ribosome biogenesis proteins Badertscher et al., 2015; Wild et al., 2010 N/A Original western blot images This paper Mendeley: https://doi.org/10.17632/sgmwr9z28b.1 Experimental Models: Cell Lines Human HeLa cells ATCC N/A Human HEK293 cells ATCC CRL-1573 NIH 3T3 mouse fibroblast cells ATCC N/A Mouse embryonic stem cells (E14) Michel Vermeulen (Radboud Institute for Molecular Life Sciences N/A Flp-In T-REx 293 Cell Line Thermo Fisher Scientific R78007 19DN mouse B cells and RPL12 mutant cells Sander et al., 2012; this paper N/A Oligonucleotides Oligonucleotides used for genome editing This paper see Generation of RPL12 Point Mutant Cell Lines via CRISPR/Cas9 Recombinant DNA pDONR221 Thermo Fisher Scientific Cat#12536017 pDEST26_FLAG and HA This paper N/A pcDNA5-FLAG and HA-RPL12-WT This paper N/A pcDNA5-FLAG and HA-RPL12-S38D This paper N/A pcDNA5-FLAG and HA-RPL12-S38A This paper N/A pFRT/TO/FLAG/HA-DEST Thomas Tuschl Addgene ID: 26360 pX330-Cas9-RPL12sgRNA This paper N/A pX330-E2A-mCherry Chu et al., 2015 N/A Software and Algorithms R studio v.1.1.4 N/A https://www.rstudio.com MaxQuant v.1.5.1.2 Cox and Mann, 2008 http://www.biochem.mpg.de/5111795/maxquant Metascape Tripathi et al., 2015 http://metascape.org/ Bcl2Fastq v.2.16.0.10 Illumina https://support.illumina.com/sequencing/ sequencing_software/bcl2fastq-conversionsoftware.html Flexbar v.2.5 Roehr et al., 2017 https://github.com/seqan/flexbar/wiki collapse_reads.pl script Jens, 2016 https://github.com/marvin-jens/clip_analysis STAR aligner v.2.4.2a, v.2.5.3a Dobin et al., 2013 https://github.com/alexdobin/STAR DESeq2 v.1.18.1 Love et al., 2014 https://bioconductor.org/packages/release/ bioc/html/DESeq2.html (Continued on next page) Molecular Cell 72, 1–15.e1–e9, October 4, 2018 e2

Techniques:

Journal: Molecular cell

Article Title: Phosphorylation of the Ribosomal Protein RPL12/uL11 Affects Translation during Mitosis.

doi: 10.1016/j.molcel.2018.08.019

Figure Lengend Snippet: Figure 4. Effect of RPL12/uL11 pS38 on Global Protein Synthesis (A) Conservation and phosphorylation motif of RPL12/uL11 S38. (B) Phosphorylation of S38 during the cell cycle in published phosphoproteomics data. (C) Experimental design for flow cytometric analysis of mitotic translation in HEK293 cells transiently expressing FLAG and HA-tagged RPL12/uL11. (D) Monitoring global protein synthesis in interphase and mitosis. Left: representative FACS result of cells expressing FLAG and HA RPL12/uL11 WT. Only cells expressing tagged RPL12/uL11 were gated, and the corresponding cell population was further analyzed by dual staining for phospho-H3 S10 and AHA-labeled proteins. Global protein synthesis was monitored by AHA incorporation (y axis) in interphase and mitosis (based on H3 pS10 staining, x axis). Right: each dot represents median AHA intensity calculated from the interphase or mitotic cell population in an independent experiment. The results from three independent experiments are shown. As controls, methionine (Met) incorporation into proteins instead of AHA and protein production in the presence of cycloheximide (CHX) were also monitored. (E) Mitotic index determined by flow cytometric analysis of H3 pS10-positive cells expressing tagged RPL12/uL11. Data are represented as mean ± SD. The p values were calculated using paired Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 3P for B cells This paper ProteomeXchange: PXD009267 pSILAC in B cells This paper ProteomeXchange: PXD009276 PRM assay in B cells This paper ProteomeXchange: PXD010029 RNA-seq and ribo-seq datasets This paper GEO: GSE112187 Human UniprotKB/Swiss-Prot data base (Human UniProt 2014-10) N/A https://www.uniprot.org/proteomes/ Mouse UniprotKB/Swiss-Prot data base (Mouse UniProt 2014-10) N/A https://www.uniprot.org/proteomes/ Riboproteome data Reschke et al., 2013 N/A Ribosome profiling data (mitosis versus S phase) Stumpf et al., 2013 N/A Protein complex annotation data (CORUM downloaded Jan/2017) Ruepp et al., 2010 https://mips.helmholtz-muenchen.de/corum/ STRING protein interaction database Szklarczyk et al., 2017 https://string-db.org/ RNAi data for ribosome biogenesis proteins Badertscher et al., 2015; Wild et al., 2010 N/A Original western blot images This paper Mendeley: https://doi.org/10.17632/sgmwr9z28b.1 Experimental Models: Cell Lines Human HeLa cells ATCC N/A Human HEK293 cells ATCC CRL-1573 NIH 3T3 mouse fibroblast cells ATCC N/A Mouse embryonic stem cells (E14) Michel Vermeulen (Radboud Institute for Molecular Life Sciences N/A Flp-In T-REx 293 Cell Line Thermo Fisher Scientific R78007 19DN mouse B cells and RPL12 mutant cells Sander et al., 2012; this paper N/A Oligonucleotides Oligonucleotides used for genome editing This paper see Generation of RPL12 Point Mutant Cell Lines via CRISPR/Cas9 Recombinant DNA pDONR221 Thermo Fisher Scientific Cat#12536017 pDEST26_FLAG and HA This paper N/A pcDNA5-FLAG and HA-RPL12-WT This paper N/A pcDNA5-FLAG and HA-RPL12-S38D This paper N/A pcDNA5-FLAG and HA-RPL12-S38A This paper N/A pFRT/TO/FLAG/HA-DEST Thomas Tuschl Addgene ID: 26360 pX330-Cas9-RPL12sgRNA This paper N/A pX330-E2A-mCherry Chu et al., 2015 N/A Software and Algorithms R studio v.1.1.4 N/A https://www.rstudio.com MaxQuant v.1.5.1.2 Cox and Mann, 2008 http://www.biochem.mpg.de/5111795/maxquant Metascape Tripathi et al., 2015 http://metascape.org/ Bcl2Fastq v.2.16.0.10 Illumina https://support.illumina.com/sequencing/ sequencing_software/bcl2fastq-conversionsoftware.html Flexbar v.2.5 Roehr et al., 2017 https://github.com/seqan/flexbar/wiki collapse_reads.pl script Jens, 2016 https://github.com/marvin-jens/clip_analysis STAR aligner v.2.4.2a, v.2.5.3a Dobin et al., 2013 https://github.com/alexdobin/STAR DESeq2 v.1.18.1 Love et al., 2014 https://bioconductor.org/packages/release/ bioc/html/DESeq2.html (Continued on next page) Molecular Cell 72, 1–15.e1–e9, October 4, 2018 e2

Techniques: Phospho-proteomics, Expressing, Staining, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

doi: 10.1073/pnas.1619266114

Figure Lengend Snippet: VP4 proteomic network reveals cytoskeleton-binding proteins that modulate RV infection. (A) Interactome of the bait viral protein (VP4) and high-confidence host binding proteins. Solid lines with arrowheads represent interactions identified in this study. The width of lines corresponds to the strength of interaction detected in the IP–MS experiment. Dotted lines represent publicly curated protein–protein interactions. The proteins studied in this paper are highlighted by yellow nodes. (B) Pie chart of PANTHER functional classification of VP4-interacting proteins shown in A. (C) HEK293 cells were transfected with indicated siRNA for 48 h and infected with simian RV RRV strain at MOI = 1 for 24 h. Levels of RV replication were measured by RT-qPCR examining the expression of viral gene VP7, normalized to that of GAPDH. The genes studied in this paper are outlined by red boxes. (D) Same experiment as in C except that viral gene NSP5 was measured instead of VP7 by RT-qPCR and virus titer in the supernatants was determined by a focus-forming unit (FFU) assay. For C and D, experiments were repeated at least five times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (**P ≤ 0.01; ***P ≤ 0.001).

Article Snippet: Human embryonic kidney fibroblast HEK293 cells (CRL-1573) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Binding Assay, Infection, Protein-Protein interactions, Functional Assay, Transfection, Quantitative RT-PCR, Expressing, Virus

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

doi: 10.1073/pnas.1619266114

Figure Lengend Snippet: Drebrin deficiency results in enhanced RV infection. (A) Lysates of HEK293 cells stably expressing a GFP-tagged RRV VP4 were stimulated with or without doxycycline (Dox, 1 μg/mL) for 24 h, then subjected to IP using α-GFP antibody, and analyzed with Western blot using indicated antibodies. Bottom panels are 10% input. (B) HEK293 cells were infected with RRV at MOI = 3 for 24 h and analyzed by confocal microscopy for the localization of VP4 (green), DBN1 (red), actin (phalloidin, white), and nucleus (DAPI, blue). Colocalization (yellow) is highlighted by white arrowheads. Panels are single z slices. (Scale bar, 8 μm.) (C) WT and two individual clones of DBN1 knockout (KO) HEK293 cells were infected with RRV at MOI = 1 for 24 h. Viral gene NSP5 expression was measured by RT-qPCR and normalized to that of GAPDH (Left). Virus particles in the supernatants were titrated by an FFU assay (Right). (D) DBN1 KO HEK293 cells were transfected with pCMV6-entry empty vector (ev), and Flag-tagged full-length DBN-A or DBN-E, and infected with RRV at MOI = 1 for 24 h. Viral gene NSP5 level was measured by RT-qPCR and normalized to that of GAPDH (Left) and lysates were harvested for Western blot using indicated antibodies (Right). (E) WT and DBN1 KO HEK293 cells were transfected with RV double-layered particles (DLPs) for 16 h and NSP5 expression was measured by RT-qPCR. (F) WT and DBN1 KO HEK293 cells were infected with RRV (MOI = 50) and then incubated at either 4 °C for 1 h or incubated at 4 °C for 1 h and then shifted to 37 °C for an additional 1 h. The level of viral gene NSP5 was measured by RT-qPCR and normalized to that of GAPDH, as an indicator of input viral genomes. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (**P ≤ 0.01; ***P ≤ 0.001; n.s., not significant).

Article Snippet: Human embryonic kidney fibroblast HEK293 cells (CRL-1573) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Infection, Stable Transfection, Expressing, Western Blot, Confocal Microscopy, Clone Assay, Knock-Out, Quantitative RT-PCR, Virus, Transfection, Plasmid Preparation, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

doi: 10.1073/pnas.1619266114

Figure Lengend Snippet: Loss of drebrin leads to enhanced endocytosis of dynamin-dependent cargo. (A) WT and DBN1 KO HEK293 cells were incubated with FITC-conjugated transferrin (Tfn, 20 μg/mL) and fixed at indicated time points for confocal microscopy to examine intracellular transferrin (green) and nucleus (DAPI, blue). (Scale bar in panels and single z slices, 20 μm.) (B) WT and DBN1 KO HEK293 cells were incubated with FITC-conjugated cholera toxin subunit B (CTxB, 10 μg/mL) and fixed at indicated time points for confocal microscopy for internalized toxin (green) and nucleus (DAPI, blue). (Scale bar in panels and single z slices, 20 μm.) (C) WT and DBN1 KO HEK293 cells were infected with human adenovirus serotype 5 (HAdV5) or simian vacuolating virus 40 (SV40) at MOI = 0.1 for 1 h. Expression levels of hexon (HAdV5) and large T antigen (SV40) were measured by RT-qPCR and normalized to that of GAPDH. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (***P ≤ 0.001).

Article Snippet: Human embryonic kidney fibroblast HEK293 cells (CRL-1573) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Incubation, Confocal Microscopy, Infection, Virus, Expressing, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

doi: 10.1073/pnas.1619266114

Figure Lengend Snippet: Drebrin inhibits dynamin-2–mediated endocytosis via interaction with cortactin. (A) WT and DBN1 KO HEK293 cells were treated with either vehicle control (DMSO) or dynasore (100 μM) for 30 min before RRV infection (MOI = 1). Total RNA was harvested at 1 hpi and NSP5 level was measured by RT-qPCR and normalized to that of GAPDH. (B) WT HEK293 cells were transfected with indicated siRNA for 48 h before infection with RRV at MOI = 1 for 1 h. Viral NSP5 level was measured by RT-qPCR and normalized to that of GAPDH. Dotted line denotes NSP5 level of cells transfected with ctrl siRNA (set at 0.984); cortactin (encoded by CTTN) is outlined by a red box. (C) DBN1 KO HEK293 cells were transfected with indicated siRNA (gelsolin, encoded by GSN) for 48 h and infected with RRV at MOI = 1 for 1 h. The level of viral NSP5 was measured by RT-qPCR and normalized to that of GAPDH. (D) HEK293 cells were transfected with the first siRNA for 48 h and then transfected with second siRNA for an additional 48 h before RRV infection (MOI = 1) for 1 h. The input viral genome was measured by NSP5 levels using RT-qPCR and normalized to that of GAPDH. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., not significant).

Article Snippet: Human embryonic kidney fibroblast HEK293 cells (CRL-1573) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Control, Infection, Quantitative RT-PCR, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Drebrin restricts rotavirus entry by inhibiting dynamin-mediated endocytosis

doi: 10.1073/pnas.1619266114

Figure Lengend Snippet: N-terminal domain of drebrin localizes to the actin cytoskeleton and coprecipitates with cortactin. (A) DBN1 KO HEK293 cells stably expressing indicated GFP-tagged DBN1 constructs or control EGFP were analyzed by confocal microscopy for the localization of DBN1 (green), actin (red), and nucleus (DAPI, blue). Colocalization (yellow) is highlighted by white arrowheads. (Full-length: DBN isoforms A and E; N terminus: amino acid 1–366; middle region: 233–317; C terminus: 319–707). (Scale bar in panels and single z slices, 40 μm.) (B) DBN1 KO HEK293 cells stably expressing indicated GFP-tagged DBN1 constructs or control EGFP were subject to IP using α-GFP antibody and analyzed by Western blot using indicated antibodies. The IP band intensities were normalized to endogenous CTTN levels in IP input and compared with that of DBN-A (lane 1), which was set as 1.00. Bottom panels are 10% input. (C) Reconstituted DBN1 KO HEK293 cells were infected with RRV at MOI = 1 for 24 h and examined by RT-qPCR for viral NSP5 expression, normalized to that of GAPDH. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (***P ≤ 0.001).

Article Snippet: Human embryonic kidney fibroblast HEK293 cells (CRL-1573) were obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Stable Transfection, Expressing, Construct, Control, Confocal Microscopy, Western Blot, Infection, Quantitative RT-PCR

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 2. [ 3H]Kainate binding and functional assessment by patch-clamp recording of GluR6-binding site mutants. A, Saturation analysis of [ 3H]kainate binding to GluR6-wt ex- pressedinHEK293cellsyieldedaKDof36nMandaBmaxof3.8pmol/mgprotein.Specificbinding wasdeterminedinthepresence(nonspecific)andabsence(total)of1mMglutamate.B,Specific binding of 100 nM [ 3H]kainate to GluR6-wt, R523G, T690G, E738G, and E738D (60 g of pro- tein). Western blots confirmed similar amounts of C-GluR6 immunoreactivity in 25 g of pro- tein used for the binding assays (inset). C, Outside-out patches were pulled from HEK293 cells expressing GluR6-wt, R523G, T690G, or E738G, voltage clamped at 70 mV, and tested by ultrafast application of 3 or 30 mM glutamate (GLU). For GluR6-wt, 3 mM glutamate was satu- rating and elicited large peak currents (2.8 0.4 nA). No currents were detected in patches from cells expressing R523G, T690G, or E738G. Application of 3 or 30 mM glutamate demon- stratesthatE738Dreceptorsarefunctional,whereas3mMglutamateisnolongersaturatingfor this mutant. D, Dose–response curve showing the rightward shift of the EC50 value ( 100- fold) for the mutation E738D compared with GluR6-wt in peak response to glutamate.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Binding Assay, Functional Assay, Patch Clamp, Western Blot, Expressing, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure 3. Cell-surface expression of the GluR6-binding site mutants. A, Confocal images of extracellular N-GluR6 (green) and intracellular C-GluR6 (red) immunofluorescence staining in HEK293 cells expressing GluR6-wt and GluR6-binding site mutants. N-GluR6 staining before cell membrane permeabilization demonstrates robust surface expression of GluR6-wt and E738D. In contrast, the absence of N-GluR6 staining indicates that R523G, T690G, and E738G are not present at the cell surface. C-GluR6 staining was evident for GluR6-wt and GluR6 mutants after permeabilization. Staining of nontransfected controls was negative. B,PrimaryculturesofratastrocytestransfectedwithGluR6-wtorT690GwerestainedforN-andC-GluR6asabove.C,Biotinylated cell-surfaceproteins(50g)wereprecipitatedwithNeutrAvidinbeads.Onlytraceamounts,ifany,ofR523G,T690G,andE738G werefoundatthecellsurface.GluR6-wtandE738Dwerebothisolatedinthebiotinylatedfraction.Thepercentageofcell-surface expressionofimmunoreactiveGluR6wasdeterminedbydensitometryusing10goftotalprotein.Theblotisrepresentativeof three separate experiments. IB, Immunoblot; IP, immunoprecipitate. D, Biotinylation studies, using GluR1-wt as a control, re- vealed that a homologous GluR1-binding site mutant, R499G, also fails to reach the cell surface.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Expressing, Binding Assay, Immunofluorescence, Staining, Membrane, Western Blot, Control, Mutagenesis

Journal: Journal of Neuroscience

Article Title: Glutamate Receptor Trafficking: Endoplasmic Reticulum Quality Control Involves Ligand Binding and Receptor Function

doi: 10.1523/jneurosci.4573-04.2005

Figure Lengend Snippet: Figure4. ERretentionandglycosylationstateofGluR6-bindingsitemutants.A,ConfocalimagesofintracellularC-GluR6(red) immunofluorescence staining in HEK293 cells coexpressing the EYFP-ER marker and GluR6-wt or GluR6-binding site mutants. GluR6-wt and E738D demonstrate robust surface staining that is not colocalized with EYFP-ER. In contrast, R523G, T690G, and E738G are not present at the cell surface and are colocalized entirely with EYFP-ER. The merged images show the overlapping localization of C-GluR6 and EYFP-ER markers. Identical results were obtained with antibodies to other ER markers, calnexin and calreticulin. B, Representative confocal images of the GluR6-wt (top) or the GluR6-binding-deficient mutant R523G (bottom) cotransfectedwiththeEYFP-Golgimarker.Themergedimageofimmunofluorescencestainingdemonstratesthelackofcolocal- ization of R523G (red) and the EYFP-Golgi marker (green). C, Triton X-100 (0.5%) solubilized lysates of HEK293 cells transfected withGluR6-wtorGluR6-bindingsitemutantsweresubjectedtoEndoHdigest.Westernblotanalysisofcontrol(Gly)andEndoH- digested(Cut)samples(15–30gofprotein)revealsthatR523G,T690G,andE738GareentirelyEndoHsensitive.Incontrast,the majorityofGluR6-wtandE738DareEndoHinsensitive.D,ParalleldigestsdemonstratePNGaseFandEndoHdeglycoslateR523G, T690G,andE738Gtothesameextent,confirmingtheirimmatureN-linkedglycosylationstate.Theblotsarerepresentativeofat least three separate experiments.

Article Snippet: Human embryonic kidney 293 (HEK293) fibroblasts (CRL 1573; American Type Culture Collection, Manassas, VA) were cultured in minimal essential medium supplemented with 10% fetal bovine serum and 2 mM glutamine (Invitrogen, Gaithersburg, MD) and maintained at 37°C in a 5% CO2 environment.

Techniques: Immunofluorescence, Staining, Marker, Binding Assay, Mutagenesis, Transfection