endoglycosidaseh  (New England Biolabs)

Journal: PLoS ONE

Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

doi: 10.1371/journal.pone.0207948

Figure Lengend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

Article Snippet: Endoglycosidase H (endo H) digestion and Peptide-N-Glycosidase F (PNGase F) (New England Biolabs) digestion were performed according to the published procedure [ ].

Techniques: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

Journal: PLoS ONE

Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

doi: 10.1371/journal.pone.0207948

Figure Lengend Snippet: BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p

Article Snippet: Endoglycosidase H (endo H) digestion and Peptide-N-Glycosidase F (PNGase F) (New England Biolabs) digestion were performed according to the published procedure [ ].

Techniques: Stable Transfection, Expressing, Cell Culture, SDS Page, Western Blot, Positive Control, Fluorescence, Incubation, Quantitation Assay

Journal: Journal of Virology

Article Title: A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

doi: 10.1128/JVI.75.13.5762-5771.2001

Figure Lengend Snippet: Identification of FV Env subdomains by different antisera. Cell or virus lysates of 293T cells transfected with the Gag/Pol-expressing FV vector pMH62 (Gag/Pol), wild-type FV Env expression construct pcHFE EM02 (Env), or empty expression vector (pCDNA3.1) as indicated were analyzed by RIPA or Western blotting. The identity of each FV protein is indicated. The gp48 TM protein often comigrates with an immunoreactive cellular protein present in negative controls. (A) Schematic outline of the transfected expression constructs. SFFV, spleen focus-forming virus; bgH pA, bovine growth hormone polyadenylation site. (B) RIPA of cells labeled for 20 h using polyclonal rabbit sera specific for Gag (α-Gag), SP Env /SU (α-SP/SU), or SP Env (α-SP). A longer exposure of the lower part of the SDS–10% polyacrylamide gel with the cellular lysates is shown separately, and an autoradiogram of metabolically labeled purified FV particles separated by SDS-PAGE is shown to the right. (C) Western blot analysis (SDS-PAGE [12% gel]) of cellular lysates and purified FV particles using polyclonal rabbit sera specific for Gag (α-Gag) or SP Env (α-SP). (D) Western blot analysis (SDS-PAGE [12% gel]) of equilibrium sedimentation gradient fractions using polyclonal rabbit sera specific for Gag (α-Gag) and SP Env (α-SP). (E) Pulse-chase analysis of FV Env maturation. Transfected 293T cells were pulse-labeled for 30 min and then chased for different time periods as indicated at the top with fresh growth medium containing an excess of cold methionine and cysteine. Equal cell lysate samples were immunoprecipitated with FV-specific antisera as indicated to the left. Equivalent aliquots of the protein A eluates were incubated with glycosidases as indicated at the top prior to separation by SDS-PAGE (7.5% gel). Identities of the different forms (g [fully glycosylated], h [endo H resistant] and p [N-deglycosylated]) of gp130 Env (solid arrows), gp80 SU (shaded arrows), and gp48 TM (open arrows) are indicated. The bands marked with asterisks at the 1- and 3-h time point after immunoprecipitation with anti-SP/SU antiserum and PNGase F treatment represent the fully N-deglycosylated form of SU running only slightly faster than the fully glycosylated form of TM.

Article Snippet: For glycosidase treatment, protein A-Sepharose eluates were denatured by boiling in 0.5% SDS–1% β-mercaptoethanol and subsequently incubated with endoglycosidase H (endo H) peptide N -glycosidase or (PNGase F) in the appropriate incubation buffer as suggested by the manufacturer (New England Biolabs) prior to loading on gels for SDS-polyacrylamide gel electrophoresis (PAGE).

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Western Blot, Labeling, Metabolic Labelling, Purification, SDS Page, Sedimentation, Pulse Chase, Immunoprecipitation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions *

doi: 10.1074/jbc.M109.044362

Figure Lengend Snippet: Implication of CD44 in coat recovery. A , Western blots of CD44 using HCAM antibody with and without endoglycosidase F treatment. B and C , coat thickness measured by the particle exclusion assay. In B , after treatment with 5 units/ml Streptomyces hyaluronidase

Article Snippet: In some cases, cell lysates were pretreated with endoglycosidase F (New England Biolabs).

Techniques: Western Blot, Exclusion Assay

Journal: Molecular Biology of the Cell

Article Title: The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

doi: 10.1091/mbc.E07-08-0806

Figure Lengend Snippet: The pH-responsive Rim101 pathway is involved in lipid asymmetry signaling. (A and B) Cells carrying single or double mutations were grown in YPD medium. Total proteins (1.7 μg) prepared from each culture were incubated with Endo H, separated by

Article Snippet: Proteins (5 μl) in 1× SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and a trace amount of bromophenol blue containing 5% 2-mercaptoethanol) were diluted with 40 μl 0.05 M sodium citrate (pH 5.5) containing 1 mM phenylmethylsulfonyl fluoride, and treated at 37°C for 1 h with 1000 U of recombinant endoglycosidase H (Endo H; New England Biolabs, Beverly, MA).

Techniques: Incubation