Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , We designed different 1059 SOSIP variants based on published studies and current knowledge. Env changes associated with each variant are shown and highlighted in green relative to the WT (red). b , Western blot of different 1059 SOSIP variants expressed in 293F cells for 4 days and analyzed directly from cell supernatants. We detected the SOSIP Env proteins using 1:10,000 dilution of serum of PLWH + 0.5μg/ml of JR52 antibody, which recognizes the D7324 epitope, followed by anti-human (1:10,000 dilution) + anti-mouse (1:20,000 dilution), both conjugated to horseradish peroxidase (HRP). Lanes: 1, ladder; 2, HIV-1 AD8 gp120 control; 3, TPA WT; 4, TPA P22A (contains amino acid Alanine at position 22); 5, DS; 6, V5.2.8a; 7, v4.1. TPA, signal peptide of the Tissue Plasminogen Activator. TPA-1059 SOSIP exhibited the highest expression levels and was selected for further characterization in all studies described in the main text. c , Comparison of size exclusion chromatography (SEC) profile of 1059 and BG505 SOSIP v6 trimers (both contained the signal peptide derived from the TPA). d , ELISA of 17b antibody binding to 1059 and BG505 SOSIPs in the presence and absence of sCD4. e , SEC profiles of 1059-SOSIP and BG505-SOSIP preparations used for determining structures of the unliganded Envs. The dashed vertical lines indicate the fractions that were pooled for downstream studies.

Article Snippet: After 1 hour and 30 minutes incubation, wells were washed 6 times and 1:5000 dilution of horseradish peroxidase (HRP)-conjugated donkey anti-human IgG (FC specific; Jackson ImmunoResearch Laboratories, West Grove, PA) was added in blocking solution to each well and the plate was incubated for 1 hour at RT.

Techniques: Variant Assay, Western Blot, Control, Expressing, Comparison, Size-exclusion Chromatography, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Journal of Diabetes Research

Article Title: Precedence of Bone Loss Accompanied with Changes in Body Composition and Body Fat Distribution in Patients with Type 2 Diabetes Mellitus

doi: 10.1155/2023/6753403

Figure Lengend Snippet: Demographic data of 596 patients with T2DM.

Article Snippet: The blood samples were collected after fasting for more than 8 h. We measured the levels of fasting plasma glucose, glycosylated hemoglobin A1c (HbA1c) (variant II glycosylated hemoglobin analyzer, Bio-Rad, high-performance liquid chromatography), total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) (Siemens ADVIA 2400 automatic biochemical analyzer).

Techniques:

Journal: Journal of Diabetes Research

Article Title: Precedence of Bone Loss Accompanied with Changes in Body Composition and Body Fat Distribution in Patients with Type 2 Diabetes Mellitus

doi: 10.1155/2023/6753403

Figure Lengend Snippet: Linear regression analysis of body composition index, L 1-4 BMD, and FNBMD. Note: adjusted: age, sex, course of T2DM, chronic complications of T2DM, BMI, FBG, HbA1c, TG, LDL-C, HDL-C, SBP, DBP, and medication history. FMI: fat mass index; MMI: muscle mass index; M/F: muscle/fat mass ratio; TFMI: trunk fat mass index; ASMI: appendicular skeletal muscle mass index; A/T: appendicular skeletal muscle mass/trunk fat mass ratio.

Article Snippet: The blood samples were collected after fasting for more than 8 h. We measured the levels of fasting plasma glucose, glycosylated hemoglobin A1c (HbA1c) (variant II glycosylated hemoglobin analyzer, Bio-Rad, high-performance liquid chromatography), total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) (Siemens ADVIA 2400 automatic biochemical analyzer).

Techniques:

Journal: Journal of Diabetes Research

Article Title: Precedence of Bone Loss Accompanied with Changes in Body Composition and Body Fat Distribution in Patients with Type 2 Diabetes Mellitus

doi: 10.1155/2023/6753403

Figure Lengend Snippet: Binary logistic regression analysis of body mass index, body composition index, and FNBMD reduction. Note: adjusted: age, sex, course of T2DM, chronic complications of T2DM, BMI, FBG, HbA1c, TG, LDL-C, HDL-C, SBP, DBP, and medication history. FMI: fat mass index; MMI: muscle mass index; M/F: muscle/fat mass ratio; TFMI: trunk fat mass index; ASMI: appendicular skeletal muscle mass index; A/T: appendicular skeletal muscle mass/trunk fat mass ratio.

Article Snippet: The blood samples were collected after fasting for more than 8 h. We measured the levels of fasting plasma glucose, glycosylated hemoglobin A1c (HbA1c) (variant II glycosylated hemoglobin analyzer, Bio-Rad, high-performance liquid chromatography), total cholesterol, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) (Siemens ADVIA 2400 automatic biochemical analyzer).

Techniques:

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a Typical image examples of CR and CS gastric cancer patients who received NAC. The volcano map ( b ) and heatmap ( c ) of the most upregulated/downregulated differentially expressed proteins in CR and CS tumor tissues. The qRT–PCR ( d , n = 22) and western blotting ( e , n = 4) results showed the expression of CRABP2 in tumor tissues of CR and CS patients. The CCK-8 results of AGS cells treated with OXA ( f ) or fluorouracil ( g ) after knockdown/overexpression of CRABP2. h The relative expression of CRABP2 in parental and OXA-resistant GC cells by qRT–PCR. i The expression of CRABP2 from the TCGA database (tumor = 335; normal = 26). Mann–Whitney U test (Wilcoxon rank sum test). The outlier values (median±2 IQR) and extreme values (median ± 3.5 IQR) were excluded. IQR: interquartile range. The expression of CRABP2 in tumor tissues and adjacent normal tissues of GC patients by qRT–PCR ( j , n = 44) and western blotting ( k , n = 12). l Typical IHC images of CRABP2-positive and CRABP2-negative tumor tissues. m The correlation curves between CRABP2 expression and OS of GC patients ( n = 488). (CR chemotherapy resistance, CS chemotherapy sensitivity, OXA oxaliplatin, C tumor tissue, N normal tissues, IHC immunohistochemistry, OS overall survival). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Knockdown, Over Expression, MANN-WHITNEY, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: Correlation between CRABP2 expression and the clinicopathological characteristics of GC patients.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: Univariate and multivariate analysis of factors associated with overall survival in GC patients.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a After knocking down CRABP2, the viability of AGS-OXA and HGC-27-OXA cells was determined under different concentrations of OXA. b After knocking down CRABP2, the colony forming ability of AGS-OXA and HGC-27-OXA cells was determined in the absence or presence of OXA (concentration: 2.0 μM). c The cell apoptosis results of AGS-OXA and HGC-27-OXA cells after knocking down CRABP2 in the presence or absence of OXA (concentration: 2.0 μM) by flow cytometry. d In the presence of different concentrations of OXA, AGS and HGC-27 cells overexpressing CRABP2 were determined by cell viability assay. e In the presence or absence of OXA (concentration: AGS 0.2 μM, HGC-27 0.4 μM), AGS and HGC-27 cells overexpressing CRABP2 were determined by the colony forming assay. f In the absence or presence of OXA (concentration: AGS 0.2 μM, HGC-27 0.4 μM), the cell apoptosis results of AGS and HGC-27 cells overexpressing CRABP2 were determined by flow cytometry. All experiments were performed in three replicates. The data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Concentration Assay, Flow Cytometry, Viability Assay

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a Total proteins from Flag-CRABP2 plasmid-transfected AGS cells were separated via SDS–PAGE. PARKIN and BAX were identified by LC/LC–MS in the CRABP2 protein complex. b Mutual interactions of BAX/PARKIN and Flag-CRABP2 were verified by the Co-IP assay. c The BAX and PARKIN proteins were pulled down by GST-CRABP2 fusion protein-bound beads by SDS–PAGE analysis. d The interaction between BAX and CRABP2 and between PARKIN and CRABP2 was verified by co-IP assay and western blotting. e After interference with CRABP2 in AGS cells, the binding ability of BAX to PARKIN decreased. f After BAX interference in AGS cells, the binding ability of CRABP2 to PARKIN was not affected. g After interference with PARKIN in AGS cells, the binding ability of CRABP2 to PARKIN was not affected. The immunofluorescence assay showed the subcellular localizations (red frame) of CRABP2/BAX/PARKIN in AGS ( h ) and HGC-27 ( i ) cells. AGS cells overexpressing ( j ) or knocking down ( k ) CRABP2 were treated with cycloheximide (CHX) for 0, 2, 4, 6, 8, or 10 h. The expression of Bax was detected by western blotting. All experiments were performed in three replicates. * P < 0.05, ** P < 0.01, *** P < 0.001. (IB immunoblotting, IP immunoprecipitation, WCL whole cell lysates).

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Plasmid Preparation, Transfection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, Immunofluorescence, Expressing, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a After adding MG-132 to AGS cells with knockdown or overexpression of CRABP2, Co-IP of Bax was performed, and ubiquitin was detected by western blotting. b GC cells with CRABP2 knockdown or overexpression were transfected with HA-labeled ubiquitinated plasmids of different sites, and Co-IP experiments of HA were performed. After adding OXA to AGS/HGC-27 cells, the activities of caspase 9 ( c ) and caspase 12 ( d ) were detected. e After adding OXA to AGS/HGC-27 cells, the expression of CC3 was detected. f After adding OXA to AGS/HGC-27 cells, the expression of DNA damage/repair-related proteins was examined by western blotting. g After AGS/HGC-27 cells were treated with OXA, the cytoplasm and mitochondria were separated, and the expression of BAX was examined. h The activities of caspase 9 were detected after CRABP2 knockdown/overexpression in AGS/HGC-27 cells. i The expression of CC3 was examined after CRABP2 knockdown/overexpression and OXA added to AGS/HGC-27 cells. j After adding OXA to AGS/HGC-27 cells and knocking down/overexpressing CRABP2, the expression of DNA damage/repair-related proteins was examined by western blotting. k , l After AGS/HGC-27 cells with CRABP2 knockdown or overexpression were treated with OXA, the cytoplasm and mitochondria were separated, and the expression of BAX was examined. (OXA oxaliplatin, CC3 cleaved caspase 3, TOMM40 translocase of outer mitochondrial membrane 40).

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Knockdown, Over Expression, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Western Blot, Transfection, Labeling, Expressing, Membrane

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a After knocking down CRABP2/ BAX and adding OXA (concentration: AGS 0.2 μM, HGC-27 0.4 μM), the percentage of apoptotic cells was examined by flow cytometry. b After knocking down CRABP2/ BAX and adding OXA (concentration: AGS 0.2 μM, HGC-27 0.4 μM), the activity of cleaved caspase 9 was examined. c After knocking down CRABP2/ BAX and adding OXA (concentration: AGS 0.2 μM, HGC-27 0.4 μM), the expression of cleaved caspase 3/BAX/CRABP2 was examined by western blotting. d Schematic diagram of the experimental procedures of the cell-derived xenograft model in nude mice. e Photo showing tumor formation in the four groups of nude mice. Tumor weight ( f ) and tumor volumes ( g ) of four groups of nude mice. h The expression of CRABP2 and BAX in tumors from four groups of nude mice by western blotting. i HE and CRABP2 IHC staining of tumors in four groups of nude mice. All experiments were performed in three replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Concentration Assay, Flow Cytometry, Activity Assay, Expressing, Western Blot, Derivative Assay, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a After adding 5AZ, Bobcat339, DZNEP, TSA, or SAHA to AGS and HGC-27 cells, the mRNA expression changes in CRABP2 were detected. The methylation ( b ) and hydroxymethylation ( c ) levels of OXA-resistant GC cells and parental GC cells were examined. d The expression and activity of DNA methylase, demethylase, and hydroxymethylase from OXA-resistant and parental GC cells were examined by western blotting. e The activity of hydroxymethylase TET1 was detected in OXA-resistant GC cells and parental GC cells. f Schematic diagram of the predicted and designed methylation sites in the promoter region of CRABP2. g ChIP experiments using a TET1 antibody were performed in OXA-resistant GC cells. h Hydroxymethylated ChIP assays were performed in OXA-resistant GC cells. After knocking down or overexpressing TET1 in OXA-resistant GC cells, the expression of CRABP2 was examined by qRT–PCR ( i ) and western blotting ( j ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Expressing, Methylation, Activity Assay, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer

doi: 10.1038/s41419-022-05299-2

Figure Lengend Snippet: a Schematic diagram of the patient-derived xenograft model of GC. b Photos of different tumor formations in the two groups of NOD-SCID mice. Tumor weight ( c ) and tumor volume ( d ) measurement in the two groups of mice. e Expression of CRABP2 and BAX in tumors from the two groups of mice, as determined by western blotting. f HE staining and CRABP2 staining of tumor slides from the two groups of mice. g Under normal physiological conditions, the synthesis and degradation of Bax in the cytoplasm are in equilibrium. When cells are treated with oxaliplatin, Bax integrates into the outer mitochondrial membrane, leading to the release of cytochrome C and cell apoptosis. In chemoresistant GC cells, in which the expression of CRABP2 in the cell is at a high level, CRABP2 can promote the ubiquitination degradation of Bax by combining Bax and Parkin, ultimately allowing the cells to survive. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, the slides were incubated with a mouse-derived CRABP2 (ProteinTech, Cat. #66468-1-Ig) primary antibody/ rabbit-derived BAX (ProteinTech, Cat. #50599-2-lg) primary antibody/ mouse-derived BAX (ProteinTech, Cat. #60267-1-Ig) primary antibody/rabbit-derived PARKIN (ProteinTech, Cat. #14060-1-AP) primary antibody overnight.

Techniques: Derivative Assay, Expressing, Western Blot, Staining, Membrane, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity

doi: 10.3390/ijms222112046

Figure Lengend Snippet: The Complementarity Determining Regions (CDRs) of the anti-La mab 312B confers anti-La reactivity. ( A ) Schematic representation of recombinant 312B ab constructs. Based on the murine (mature), germline, and humanized variable heavy and light chain domains of the anti-La mab 312B, different single-chain fragment variables (scFvs) were constructed and fused to the human IgG4-Fc domain to obtain recombinant murine (mature), germline, or humanized 312B ab constructs. ( B ) Recombinant 312B constructs were isolated by protein A affinity chromatography from cell culture supernatant of permanent 3T3 production cell lines. Purified proteins (312B: mature ab; gl312B: all SHMs were mutated back to the germline sequence; hu312B: CDRs of the murine 312B ab were grafted to the best fitting human framework regions) were separated by SDS-PAGE and subsequently stained with Coomassie-Brilliant Blue. ( C ) The purified 312B ab constructs were tested by SDS-PAGE/immunoblotting against human recombinant La protein (rh-La) and a mutant La version in which the three cysteine residues were mutated to alanine (TCM-La), which makes La protein insensitive to oxidation. m, protein ladder (kDa).

Article Snippet: Ab binding was detected using anti-human IgG4-horseradish peroxidase (HRP) secondary ab (Southern Biotech, BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany).

Techniques: Recombinant, Construct, Isolation, Affinity Chromatography, Cell Culture, Purification, Sequencing, SDS Page, Staining, Western Blot, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity

doi: 10.3390/ijms222112046

Figure Lengend Snippet: A single aa replacement in the germline sequence restores anti-La reactivity. The SHM causing the mutation of the D residue to a Y residue in the CDR3 region of the V H domain of the 312B ab came into our focus. A 312B IgG4 construct was cloned, in which the D in the germline region was replaced with the Y residue present in the 312B aa sequence. This mutant was termed gl312B-D > Y (germline mutant of 312B in which D is replaced by Y). ( A ) The resulting construct was expressed by permanent 3T3 production cell line. The purified ab gl312B-D > Y was analyzed by SDS-PAGE and stained with Coomassie-Brilliant Blue. ( B ) SDS-PAGE/immunoblot of recombinant human La (rh-La) and the triple cysteine mutant (TCM-La) against the gl312B-D > Y ab. m, protein ladder (kDa).

Article Snippet: Ab binding was detected using anti-human IgG4-horseradish peroxidase (HRP) secondary ab (Southern Biotech, BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany).

Techniques: Sequencing, Mutagenesis, Construct, Clone Assay, Purification, SDS Page, Staining, Western Blot, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity

doi: 10.3390/ijms222112046

Figure Lengend Snippet: Co-immunoprecipitation of native human La protein. Native human La protein present in total HeLa cell extract (as confirmed in the positive control (POS)) was co-precipitated with the respective 312B ab derivate, including the humanized 312B ab (hu312B); the germline 312B ab, in which all SHMs were mutated back to the germline sequence (gl312B); the mature, murine 312B ab (312B); and the germline ab variant of 312B, in which the aspartate residue in the V H CDR3 region was replaced by a tyrosine residue (gl312B-D > Y). Co-precipitated native human La protein was detected by SDS-PAGE/immunoblotting using the anti-La mab 5B9 and anti-mouse IgG abs-conjugated with peroxidase. POS, positive control (HeLa extract); NEG, negative control (PBS); m, protein ladder; La, human La protein; LaN, N-terminal proteolysis product of La protein.

Article Snippet: Ab binding was detected using anti-human IgG4-horseradish peroxidase (HRP) secondary ab (Southern Biotech, BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany).

Techniques: Immunoprecipitation, Positive Control, Sequencing, Variant Assay, SDS Page, Western Blot, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity

doi: 10.3390/ijms222112046

Figure Lengend Snippet: Comparison of the binding curves obtained by ELISA for the different 312B ab derivatives using the TCM-La protein as substrate. Increasing amounts of the respective ab were analyzed ranging from 0.1 to 5.000 ng/mL, including the anti-La mab as secreted from the hybridoma (anti-La 312B mab), the IgG4 construct of the humanized 312B ab (hu312B), the murine 312B ab (312B), the germline ab (gl312B), and the mutant in which the aspartate present in the V H germline sequence is replaced by a tyrosine residue (gl312B-D > Y). Each data point is the result of three estimations.

Article Snippet: Ab binding was detected using anti-human IgG4-horseradish peroxidase (HRP) secondary ab (Southern Biotech, BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Construct, Mutagenesis, Sequencing