mycobacterium szulgai  (ATCC)

Journal: Biomolecules

Article Title: A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ)

doi: 10.3390/biom14101252

Figure Lengend Snippet: Gfpt1 tm1d/tm1d mice exhibit progressive fatigable muscle weakness with disrupted NMJ morphology. ( A ) Our previously published Gftp1 tm1d/tm1d mouse model was developed with Cre/LoxP technology. This model contains two LoxP sites, which flank the seventh exon of Gfpt1 (termed Gftp1 tm1c/tm1c ). Gfpt1 tm1c/tm1c mice are crossed with the Ckm-Cre expressing mouse to produce a heterozygous mouse containing Ckm-Cre and the LoxP flanking Gfpt1, termed Gfpt1 Cre-Het . The Gfpt1 Cre-Het mice were crossed with Gfpt1 tm1c/tm1c mice to produce the Gfpt1 tm1d/tm1d mouse line. The Gfpt1 tm1d/tm1d mouse model expresses a skeletal muscle-specific truncation of Gfpt1, which is targeted for degradation. ( B ) Mice were weighed 3 times per week over 40 weeks; Gfpt1 tm1d/tm1d mice (grey line) showed no significant difference in body weight when compared to Tm1C homozygous control mice (black line). ( C ) There was a significant increase in body fat percentage in Gfpt1 tm1d/tm1d mice over the same period (EchoMRI). ( D ) Gfpt1 tm1d/tm1d mice have a progressive reduction in hanging impulse when compared to Tm1C homozygous control mice. ( E ) Repetitive grip strength measurements demonstrated that Gfpt1 tm1d/tm1d mice have increased muscle fatigability compared to Tm1C homozygous control mice. ( F ) NMJs from soleus muscle were labeled with anti-synaptic vesicle 2 to label pre-synaptic nerve terminals (red) and α-bungarotoxin to visualize AChR clusters (green). All graphs show mean ± SD, and statistical significance was determined via a two-way ANOVA. n = 8 for both Tm1C homozygous control mice and Gfpt1 tm1d/tm1d mice. *** p -value < 0.001, **** p -value < 0.0001, ns: not significant.

Article Snippet: The following antibodies were used for Western blot experiments: Gfpt1 (ProteinTech, #14132-1-AP, 1:500, Rosemont, IL, USA), Gapdh (14C10) (Cell Signalling, #2118, 1:2000, Danvers, MA, USA), O-GlcNAc (RL2) (Novus Biologicals, #NB300-524, 1:1000, Toronto, ON, Canada), Ogt (ProteinTech, #66823-1-Ig, 1:1000, Rosemont, IL, USA), Desmin (ProteinTech, #16520-1-AP, 1:1000, Rosemont, IL, USA), AChRα (ProteinTech, #10613-1-AP, 1:1000, Rosemont, IL, USA), AChRδ (C-4) (Santa Cruz, #sc-390896, 1:1000, Dallas, TX, USA), PolySia 735 (gift from Dr. Rita Gerardy-Schahn, Hannover, Germany, 1:1000), Vinculin (7F9) (Santa Cruz, #sc-73614, 1:2000, Dallas, TX, USA), Lrp4 (extracellular) (NeuroMab N207/27, #75-221, UC Davis/NIH NeuroMab Facility, Davis, CA, USA), biotinylated-succinylated wheat germ agglutinin (sWGA) (Vector Laboratories, #B-1025S-5, 1:1000, Newark, CA, USA), and IRDye ® 800CW Streptavidin (Li-Cor, #926-32230, 1:5000).

Techniques: Expressing, Control, Labeling

Journal: Biomolecules

Article Title: A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ)

doi: 10.3390/biom14101252

Figure Lengend Snippet: Gfpt1 tm1d/tm1d mice exhibit impaired Chrnd gene and AChRδ protein expression with the presence of an altered molecular weight banding pattern. ( A ) Chrnd and ( B ) Chrna1 gene expression are reduced in quadriceps isolated from 40-week-old Gfpt1 tm1d/tm1d mice normalized to reference gene expression. ( C ) A low-molecular-weight species (60 kDa) can be detected in AChRδ in Gfpt1 tm1d/tm1d skeletal muscle lysates on Western blot. ( D ) AChRδ protein levels are reduced in Gfpt1 tm1d/tm1d skeletal muscle compared to Tm1C homozygous control mice, normalized to Gapdh loading control. ( E ) Gfpt1 tm1d/tm1d mice exhibited a reduction in the ratio of the high-molecular-weight species and the low-molecular-weight species of AChRδ upon comparison to Tm1C homozygous control mice. ( F ) Provided the change in AChRδ, we examined additional AChR subunit protein levels. There was no discernable change in molecular weight with AChRα. ( G ) There was no significant change in AChRα protein levels within Gfpt1 tm1d/tm1d quadricep. ( H ) A Western blot showing quadriceps and ( I ) gastrocnemius muscle lysates from Gfpt1 tm1d/tm1d and Tm1C homozygous control following treatment with PNGase F. PNGase F treatment resulted in the migration of the 65 kDa and 60 kDa species of AChRδ species, suggesting that the lower molecular weight is hypo-glycosylated. The molecular weight species of AChRδ are labeled with 65kDa and 60kDa in green. A lower 36kDa species is labelled with yellow arrows. For in vivo experiments assessing protein levels and RNA expression, three mice were assessed ( n = 3). Original images can be found in . Graphs show mean ± SD; statistical significance was determined by Student’s t -Test. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, ns: not significant.

Article Snippet: The following antibodies were used for Western blot experiments: Gfpt1 (ProteinTech, #14132-1-AP, 1:500, Rosemont, IL, USA), Gapdh (14C10) (Cell Signalling, #2118, 1:2000, Danvers, MA, USA), O-GlcNAc (RL2) (Novus Biologicals, #NB300-524, 1:1000, Toronto, ON, Canada), Ogt (ProteinTech, #66823-1-Ig, 1:1000, Rosemont, IL, USA), Desmin (ProteinTech, #16520-1-AP, 1:1000, Rosemont, IL, USA), AChRα (ProteinTech, #10613-1-AP, 1:1000, Rosemont, IL, USA), AChRδ (C-4) (Santa Cruz, #sc-390896, 1:1000, Dallas, TX, USA), PolySia 735 (gift from Dr. Rita Gerardy-Schahn, Hannover, Germany, 1:1000), Vinculin (7F9) (Santa Cruz, #sc-73614, 1:2000, Dallas, TX, USA), Lrp4 (extracellular) (NeuroMab N207/27, #75-221, UC Davis/NIH NeuroMab Facility, Davis, CA, USA), biotinylated-succinylated wheat germ agglutinin (sWGA) (Vector Laboratories, #B-1025S-5, 1:1000, Newark, CA, USA), and IRDye ® 800CW Streptavidin (Li-Cor, #926-32230, 1:5000).

Techniques: Expressing, Molecular Weight, Gene Expression, Isolation, Western Blot, Control, High Molecular Weight, Comparison, Migration, Labeling, In Vivo, RNA Expression

Journal: Biomolecules

Article Title: A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ)

doi: 10.3390/biom14101252

Figure Lengend Snippet: Gfpt1 deficient C2C12 myotubes exhibit impaired AChRδ protein levels, with the presence of a low-molecular-weight species. ( A ) Gfpt1-deficient C2C12 myoblasts were created using a tetracycline-inducible lentiviral system. A lentivirus encoding a tetracycline repressor (TET-R) was used to infect C2C12 cells under the selection of hygromycin. Next, a scramble, shGfpt1 #1 and shGfpt1 #2 expressing transgene, was infected into Tet-R positive C2C12 cells. Cells infected with the scramble and Gfpt1-deficient transgenes were selected with puromycin. ( B ) Exposure to doxycycline activates an eGFP reporter in scramble, shGfpt1 #1 and shGfpt1 #2 stable C2C12 cells. Scale bar is 200 μm. Gfpt1 protein levels were found in Gfpt1-deficient C2C12 myoblasts upon activation with doxycycline. ( C , D ) A reduction in Gfpt1 protein levels after treatment with doxycycline in Gfpt1-deficient C2C12 myoblasts was observed. ( E ) Gfpt1-deficiency in C2C12 myotubes impairs the expression of Chrnd and ( F ) Chrna1. ( G ) Gfpt1-depleted myotubes express a lower-molecular-weight 60 kDa species of AChRδ that was not present in scramble control conditions. ( H ) A reduction in AChRδ protein levels in Gfpt1-depleted C2C12 myotubes was observed. ( I ) Densitometry was used to show a reduction in the ratio of the upper molecular weight of AChRδ and the low-molecular-weight species of AChRδ in C2C12 myotubes. ( J ) There was no significant change in AChRα protein levels between scramble and Gfpt1-deficient myotubes. For in vitro experiments, protein levels and RNA expression were analyzed by three separate experiments ( n = 3). Original images can be found in . Graphs are presented as mean ± SD, and statistical significance was determined by one-way ANOVA. ns p -value > 0.05, *** p -value< 0.001, **** p -value < 0.0001, ns: not significant.

Article Snippet: The following antibodies were used for Western blot experiments: Gfpt1 (ProteinTech, #14132-1-AP, 1:500, Rosemont, IL, USA), Gapdh (14C10) (Cell Signalling, #2118, 1:2000, Danvers, MA, USA), O-GlcNAc (RL2) (Novus Biologicals, #NB300-524, 1:1000, Toronto, ON, Canada), Ogt (ProteinTech, #66823-1-Ig, 1:1000, Rosemont, IL, USA), Desmin (ProteinTech, #16520-1-AP, 1:1000, Rosemont, IL, USA), AChRα (ProteinTech, #10613-1-AP, 1:1000, Rosemont, IL, USA), AChRδ (C-4) (Santa Cruz, #sc-390896, 1:1000, Dallas, TX, USA), PolySia 735 (gift from Dr. Rita Gerardy-Schahn, Hannover, Germany, 1:1000), Vinculin (7F9) (Santa Cruz, #sc-73614, 1:2000, Dallas, TX, USA), Lrp4 (extracellular) (NeuroMab N207/27, #75-221, UC Davis/NIH NeuroMab Facility, Davis, CA, USA), biotinylated-succinylated wheat germ agglutinin (sWGA) (Vector Laboratories, #B-1025S-5, 1:1000, Newark, CA, USA), and IRDye ® 800CW Streptavidin (Li-Cor, #926-32230, 1:5000).

Techniques: Molecular Weight, Selection, Expressing, Infection, Activation Assay, Control, In Vitro, RNA Expression

Journal: Biomolecules

Article Title: A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ)

doi: 10.3390/biom14101252

Figure Lengend Snippet: The low-molecular-weight species is detected in the NMJs isolated from Gfpt1 tm1d/tm1d muscle. ( A ) Laser microdissection was used to enrich NMJs in Gfpt1 tm1d/tm1d and Tm1C homozygous control mice. Gastrocnemius muscles were longitudinally sectioned and stained for AChR clusters. ( B ) An image at 40× magnification of AChR clusters before and after isolation. Scale bar: 500 μm. ( C ) A Western blot of NMJ and muscle isolates from Gfpt1 tm1d/tm1d and Tm1C homozygous control mice for the detection of AChRδ, Desmin, and Gapdh. Gfpt1 tm1d/tm1d NMJ isolates express an upper- and low-molecular-weight species for the AChRδ subunit, which was not observed in Tm1c homozygous control NMJ isolate. AChRδ was used to assess for NMJ-specific protein detection, while desmin and Gapdh were used to examine expression in both NMJ and muscle isolates to ensure sample purity. Original image can be found in .

Article Snippet: The following antibodies were used for Western blot experiments: Gfpt1 (ProteinTech, #14132-1-AP, 1:500, Rosemont, IL, USA), Gapdh (14C10) (Cell Signalling, #2118, 1:2000, Danvers, MA, USA), O-GlcNAc (RL2) (Novus Biologicals, #NB300-524, 1:1000, Toronto, ON, Canada), Ogt (ProteinTech, #66823-1-Ig, 1:1000, Rosemont, IL, USA), Desmin (ProteinTech, #16520-1-AP, 1:1000, Rosemont, IL, USA), AChRα (ProteinTech, #10613-1-AP, 1:1000, Rosemont, IL, USA), AChRδ (C-4) (Santa Cruz, #sc-390896, 1:1000, Dallas, TX, USA), PolySia 735 (gift from Dr. Rita Gerardy-Schahn, Hannover, Germany, 1:1000), Vinculin (7F9) (Santa Cruz, #sc-73614, 1:2000, Dallas, TX, USA), Lrp4 (extracellular) (NeuroMab N207/27, #75-221, UC Davis/NIH NeuroMab Facility, Davis, CA, USA), biotinylated-succinylated wheat germ agglutinin (sWGA) (Vector Laboratories, #B-1025S-5, 1:1000, Newark, CA, USA), and IRDye ® 800CW Streptavidin (Li-Cor, #926-32230, 1:5000).

Techniques: Molecular Weight, Isolation, Laser Capture Microdissection, Control, Muscles, Staining, Western Blot, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Neuregulin-1/ErbB4 upregulates acetylcholine receptors via Akt/mTOR/p70S6K: a study in a rat model of obstetric brachial plexus palsy and in vitro .

doi: 10.3724/abbs.2022158

Figure Lengend Snippet: Figure 2. The effects of NRG-1/ErbB4 on regulating the protein synthesis of AChR subunits in L6 myoblasts The protein levels of AChRα, AChRβ, and AChRδ were upregulated in L6 myoblasts treated with NRG-1, as revealed by western blot analysis (A). The protein levels of AChRα, AChRβ, and AChRδ were upregulated in the ErbB4-overexpressing group, as revealed by western blot analysis (B,D). The protein levels of AChRα, AChRβ, and AChRδ were downregulated in L6 myoblasts transfected with ErbB4 siRNA (C, E). The mRNA levels of AChRα, AChRβ, and AChRδ showed no significant changes in ErbB4-overexpressing (F) or ErbB4-knockdown (G) L6 myoblasts, as revealed by real-time quantitative PCR. The AChR cluster is upregulated in the ErbB4-overexpressing group and downregulated in the ErbB4-knockdown group in representative confocal images (H). Red: ErbB4; green: α-bungarotoxin; blue: nucleus. Scale bar: 20 μm. n=6. ErbB4 OV versus vector, and si-ErbB4 versus NC. *P<0.05, **P<0.01. N.S. represents no significance.

Article Snippet: The membranes were incubated with anti-AChRα antibody (1:1000; Abnova, Taipei, China), anti-AChRβ antibody (1:100; Abcam, Cambridge, UK), anti-AChRδ antibody (1:5000; Santa Cruz, Santa Cruz, USA), anti-NRG-1 (1:1000; Santa Cruz), anti-ErbB2 (1:1000; Abcam), anti-ErbB3 (1:1000; Abcam), antiErbB4 (1:1000; Genetex, Irvine, USA), anti-Akt (1:1000; Cell Signaling Technology), anti-p-Akt (1:1000; Cell Signaling Technology), anti-mTOR (1:1000; Cell Signaling Technology), anti-p-mTOR (1:1000; Cell Signaling Technology), anti-p70S6K (1:1000; Cell Signaling Technology), anti-p-p70S6K (1:1000; Cell Signaling Technology), or anti-β-actin (1:1000; Sigma-Aldrich) at 4°C overnight.

Techniques: Western Blot, Transfection, Knockdown, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Acta biochimica et biophysica Sinica

Article Title: Neuregulin-1/ErbB4 upregulates acetylcholine receptors via Akt/mTOR/p70S6K: a study in a rat model of obstetric brachial plexus palsy and in vitro .

doi: 10.3724/abbs.2022158

Figure Lengend Snippet: Figure 3. The effects of Akt on regulating the protein synthesis of AChR subunits in L6 myoblasts The ErbB4-induced increases in AChRα, AChRβ, and AChRδ protein levels were blocked in L6 myoblasts treated with MK-2206 (Akt inhibitor) but were not affected by treatment with PD98059 (ERK inhibitor) or SB203580 (p38 inhibitor) (A–D). The AChF cluster is downregulated in vector and ErbB4 OV groups treated with MK- 2206, but no changes are observed in either of the groups treated with PD98059 or SB203580 in representative confocal images (E). Green: ErbB4; blue: nucleus. Scale bar: 20 μm. n=6. ErbB4 OV versus Vector, ErbB4 OV+MK-2206 versus Vector+MK-2206, ErbB4 OV+PD98059 versus Vector +PD98059, and ErbB4 OV+SB203580 versus Vector+SB203580. **P<0.01. N.S. represents no significance.

Article Snippet: The membranes were incubated with anti-AChRα antibody (1:1000; Abnova, Taipei, China), anti-AChRβ antibody (1:100; Abcam, Cambridge, UK), anti-AChRδ antibody (1:5000; Santa Cruz, Santa Cruz, USA), anti-NRG-1 (1:1000; Santa Cruz), anti-ErbB2 (1:1000; Abcam), anti-ErbB3 (1:1000; Abcam), antiErbB4 (1:1000; Genetex, Irvine, USA), anti-Akt (1:1000; Cell Signaling Technology), anti-p-Akt (1:1000; Cell Signaling Technology), anti-mTOR (1:1000; Cell Signaling Technology), anti-p-mTOR (1:1000; Cell Signaling Technology), anti-p70S6K (1:1000; Cell Signaling Technology), anti-p-p70S6K (1:1000; Cell Signaling Technology), or anti-β-actin (1:1000; Sigma-Aldrich) at 4°C overnight.

Techniques: Plasmid Preparation

Journal: Acta biochimica et biophysica Sinica

Article Title: Neuregulin-1/ErbB4 upregulates acetylcholine receptors via Akt/mTOR/p70S6K: a study in a rat model of obstetric brachial plexus palsy and in vitro .

doi: 10.3724/abbs.2022158

Figure Lengend Snippet: Figure 6. The effects of NRG-1/ErbB4 on regulating the protein synthesis of AChR subunits via mTOR in L6 myoblasts The effects of ErbB4 on increasing AChRα, AChRβ, and AChRδ protein levels are blocked in L6 myoblasts treated with rapamycin (A,B). The effects of ErbB4 on increasing the phosphorylation ratios of p-mTOR/mTOR and p-p70S6K/p70S6K were blocked in L6 myoblasts treated with rapamycin (C,D). AChR clusters are decreased in L6 myoblasts overexpressing ErbB4 and vector with rapamycin treatment in representative confocal images (E). Green: α- bungarotoxin; blue: nucleus. n=6. ErbB4 OV versus vector, and ErbB4 OV+rapamycin versus vector+rapamycin. **P<0.01. N.S. represents no significance.

Article Snippet: The membranes were incubated with anti-AChRα antibody (1:1000; Abnova, Taipei, China), anti-AChRβ antibody (1:100; Abcam, Cambridge, UK), anti-AChRδ antibody (1:5000; Santa Cruz, Santa Cruz, USA), anti-NRG-1 (1:1000; Santa Cruz), anti-ErbB2 (1:1000; Abcam), anti-ErbB3 (1:1000; Abcam), antiErbB4 (1:1000; Genetex, Irvine, USA), anti-Akt (1:1000; Cell Signaling Technology), anti-p-Akt (1:1000; Cell Signaling Technology), anti-mTOR (1:1000; Cell Signaling Technology), anti-p-mTOR (1:1000; Cell Signaling Technology), anti-p70S6K (1:1000; Cell Signaling Technology), anti-p-p70S6K (1:1000; Cell Signaling Technology), or anti-β-actin (1:1000; Sigma-Aldrich) at 4°C overnight.

Techniques: Phospho-proteomics, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Establishment of stable cell line expressing clustered AChR via a dual lentiviral system. (A) Schematic of our stable cell line expressing clustered AChR (KL525). It is established by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, β1, δ, ϵ and the clustering protein rapsyn. (B) CMV-MCS-PGK-Puro lentiviral vectors expressing human rapsyn and the AChR α1 and β1 subunits. (C) CMV-MCS-PGK-Blasticidin lentiviral vectors expressing the human AChR δ and ϵ subunits. LTR, long terminal repeat; RRE, Rev Response Element; cPPT/CTS, central polypurine tract and the central termination; CMV, Cytomegalovirus; CHRNA1, Cholinergic Receptor Nicotinic Alpha 1 Subunit; CHRNB1, Cholinergic Receptor Nicotinic Beta 1 Subunit; CHRND, Cholinergic Receptor Nicotinic Delta Subunit; CHRNE, Cholinergic Receptor Nicotinic Epsilon Subunit; RAPSN, gene encoding rapsyn; PGK, phosphoglycerate kinase; WPRE, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.

Article Snippet: After blocked by 3% bovine serum albumin (BSA, sigma) with 0.2% Triton X-100 in 1×PBS for 1 hours, the cells were incubated with diluted serum (1:100) and AChR δ antibody (1:100, sc-390896, Santa cruz) at 4°C overnight.

Techniques: Stable Transfection, Expressing, Virus

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Stable expression of AChR subunits and rapsyn in HEK293T cells. (A) Double immunofluorescence staining with 4′,6-diamidino-2-phenylindole (DAPI, blue), AChR subunits (red) and rapsyn (red) in the stable cell line KL525. Bar, 100 μm. (B–F) mRNA expression of the human AChR subunit genes CHRNA1 , CHRNB , CHRND and CHRNE and the AChR-clustering protein gene RAPSN in KL525 cells, as measured by real-time PCR. Uninfected HEK293T cells were used as the negative control (NC). (G, H) Flow cytometric analysis and representative results of coexpression of the AChR α1 & δ subunits in KL525 cells after passage. (I) Western blot analysis of AChR α1, β1, δ, ϵ subunits and rapsyn. Uninfected HEK293T cells were used as the NC. **** P < 0.0001 compared with NC.

Article Snippet: After blocked by 3% bovine serum albumin (BSA, sigma) with 0.2% Triton X-100 in 1×PBS for 1 hours, the cells were incubated with diluted serum (1:100) and AChR δ antibody (1:100, sc-390896, Santa cruz) at 4°C overnight.

Techniques: Expressing, Double Immunofluorescence Staining, Stable Transfection, Real-time Polymerase Chain Reaction, Negative Control, Western Blot

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Detection of AChR in patients with myasthenia gravis. (A) Flow diagram showing participants in this study. The cell line stably expressing the clustered acetylcholine receptor (AChR) (KL525) identified anti-AChR antibodies in 30 of 50 (60.0%) radioimmunoprecipitation assay (RIPA)-seronegative myasthenia gravis (SNMG) patients. MG indicates myasthenia gravis, and MuSK indicates muscle-specific tyrosine kinase. (B–D) Representative results of FACS results and analysis of the KL525 score (double-positive cells for both AChR expression and serum antibody binding). The threshold KL525 score was 5.78, determined according to the mean + 2 SDs of the results from the negative controls. KL525 scores in healthy control group (HC, n = 58), in patients with positive for anti-AChR antibodies with a high RIPA titer (RIPA-High, RIPA≥8 nmol/L, n = 28), in patients with positive for anti-AChR antibodies with a low RIPA titer (RIPA-Low, 0.5 nmol/L

Article Snippet: After blocked by 3% bovine serum albumin (BSA, sigma) with 0.2% Triton X-100 in 1×PBS for 1 hours, the cells were incubated with diluted serum (1:100) and AChR δ antibody (1:100, sc-390896, Santa cruz) at 4°C overnight.

Techniques: Stable Transfection, Expressing, Radio Immunoprecipitation, Binding Assay, Control, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Comparison of clinical features of RIPA-SNMG patients, RIPA-SNMG patients with anti-AChR antibodies detected by the KL525 CBA and patients with anti-AChR antibodies detected by both the KL525 CBA & RIPA.

Article Snippet: After blocked by 3% bovine serum albumin (BSA, sigma) with 0.2% Triton X-100 in 1×PBS for 1 hours, the cells were incubated with diluted serum (1:100) and AChR δ antibody (1:100, sc-390896, Santa cruz) at 4°C overnight.

Techniques: Comparison, Crocin Bleaching Assay

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Clinical characteristics of sNMG patients with positive anti-AChR antibodies detected by the KL525 CBA.

Article Snippet: After blocked by 3% bovine serum albumin (BSA, sigma) with 0.2% Triton X-100 in 1×PBS for 1 hours, the cells were incubated with diluted serum (1:100) and AChR δ antibody (1:100, sc-390896, Santa cruz) at 4°C overnight.

Techniques:

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Establishment of stable cell line expressing clustered AChR via a dual lentiviral system. (A) Schematic of our stable cell line expressing clustered AChR (KL525). It is established by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, β1, δ, ϵ and the clustering protein rapsyn. (B) CMV-MCS-PGK-Puro lentiviral vectors expressing human rapsyn and the AChR α1 and β1 subunits. (C) CMV-MCS-PGK-Blasticidin lentiviral vectors expressing the human AChR δ and ϵ subunits. LTR, long terminal repeat; RRE, Rev Response Element; cPPT/CTS, central polypurine tract and the central termination; CMV, Cytomegalovirus; CHRNA1, Cholinergic Receptor Nicotinic Alpha 1 Subunit; CHRNB1, Cholinergic Receptor Nicotinic Beta 1 Subunit; CHRND, Cholinergic Receptor Nicotinic Delta Subunit; CHRNE, Cholinergic Receptor Nicotinic Epsilon Subunit; RAPSN, gene encoding rapsyn; PGK, phosphoglycerate kinase; WPRE, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element.

Article Snippet: The proteins were incubated with primary antibodies as follows: AChR α1 (1:500, ab221868, Abcam), AChR β1 (1:500, sc-166032, Santa cruz), AChR δ (1:500, sc-390896, Santa cruz), AChR ε (1:500, sc-376747, Santa cruz), Rapsn (1:1000, ab11423, Abcam) and β-actin (1:5000, A1978, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Virus

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Stable expression of AChR subunits and rapsyn in HEK293T cells. (A) Double immunofluorescence staining with 4′,6-diamidino-2-phenylindole (DAPI, blue), AChR subunits (red) and rapsyn (red) in the stable cell line KL525. Bar, 100 μm. (B–F) mRNA expression of the human AChR subunit genes CHRNA1 , CHRNB , CHRND and CHRNE and the AChR-clustering protein gene RAPSN in KL525 cells, as measured by real-time PCR. Uninfected HEK293T cells were used as the negative control (NC). (G, H) Flow cytometric analysis and representative results of coexpression of the AChR α1 & δ subunits in KL525 cells after passage. (I) Western blot analysis of AChR α1, β1, δ, ϵ subunits and rapsyn. Uninfected HEK293T cells were used as the NC. **** P < 0.0001 compared with NC.

Article Snippet: The proteins were incubated with primary antibodies as follows: AChR α1 (1:500, ab221868, Abcam), AChR β1 (1:500, sc-166032, Santa cruz), AChR δ (1:500, sc-390896, Santa cruz), AChR ε (1:500, sc-376747, Santa cruz), Rapsn (1:1000, ab11423, Abcam) and β-actin (1:5000, A1978, Sigma-Aldrich).

Techniques: Expressing, Double Immunofluorescence Staining, Stable Transfection, Real-time Polymerase Chain Reaction, Negative Control, Western Blot

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Detection of AChR in patients with myasthenia gravis. (A) Flow diagram showing participants in this study. The cell line stably expressing the clustered acetylcholine receptor (AChR) (KL525) identified anti-AChR antibodies in 30 of 50 (60.0%) radioimmunoprecipitation assay (RIPA)-seronegative myasthenia gravis (SNMG) patients. MG indicates myasthenia gravis, and MuSK indicates muscle-specific tyrosine kinase. (B–D) Representative results of FACS results and analysis of the KL525 score (double-positive cells for both AChR expression and serum antibody binding). The threshold KL525 score was 5.78, determined according to the mean + 2 SDs of the results from the negative controls. KL525 scores in healthy control group (HC, n = 58), in patients with positive for anti-AChR antibodies with a high RIPA titer (RIPA-High, RIPA≥8 nmol/L, n = 28), in patients with positive for anti-AChR antibodies with a low RIPA titer (RIPA-Low, 0.5 nmol/L

Article Snippet: The proteins were incubated with primary antibodies as follows: AChR α1 (1:500, ab221868, Abcam), AChR β1 (1:500, sc-166032, Santa cruz), AChR δ (1:500, sc-390896, Santa cruz), AChR ε (1:500, sc-376747, Santa cruz), Rapsn (1:1000, ab11423, Abcam) and β-actin (1:5000, A1978, Sigma-Aldrich).

Techniques: Stable Transfection, Expressing, Radio Immunoprecipitation, Binding Assay, Control, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Comparison of clinical features of RIPA-SNMG patients, RIPA-SNMG patients with anti-AChR antibodies detected by the KL525 CBA and patients with anti-AChR antibodies detected by both the KL525 CBA & RIPA.

Article Snippet: The proteins were incubated with primary antibodies as follows: AChR α1 (1:500, ab221868, Abcam), AChR β1 (1:500, sc-166032, Santa cruz), AChR δ (1:500, sc-390896, Santa cruz), AChR ε (1:500, sc-376747, Santa cruz), Rapsn (1:1000, ab11423, Abcam) and β-actin (1:5000, A1978, Sigma-Aldrich).

Techniques: Comparison, Crocin Bleaching Assay

Journal: Frontiers in Immunology

Article Title: A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis

doi: 10.3389/fimmu.2021.666046

Figure Lengend Snippet: Clinical characteristics of sNMG patients with positive anti-AChR antibodies detected by the KL525 CBA.

Article Snippet: The proteins were incubated with primary antibodies as follows: AChR α1 (1:500, ab221868, Abcam), AChR β1 (1:500, sc-166032, Santa cruz), AChR δ (1:500, sc-390896, Santa cruz), AChR ε (1:500, sc-376747, Santa cruz), Rapsn (1:1000, ab11423, Abcam) and β-actin (1:5000, A1978, Sigma-Aldrich).

Techniques: