Journal: Methods in Molecular Biology

Article Title: Monoclonal Antibodies

doi: 10.1007/978-1-62703-992-5

Figure Lengend Snippet: Fig. 1 Cloning strategy for recombinant antibodies in a single expression plasmid. ( a ) Schematic representation of a Y-shaped antibody molecule consisting of two heavy and two light chains. The antigen binding site is located at the tips of the “Y”, where the amino acid sequence is unique to the antibody and specifi cally binds antigen. ( b ) Three PCR products (linker fragment, variable light, and variable heavy) are combined by fusion PCR ( dotted red lines indicate overlap between single PCR products). The product of the fusion PCR and the plasmid are digested with NotI and AscI to allow a directed insertion into a mammalian expression plasmid backbone (Color fi gure online)

Article Snippet: Amplify the linker fragment from one of our recombinant antibodies (e.g., SI3-Flrt3, Addgene ID: 28217; http://www. addgene.com ).

Techniques: Cloning, Recombinant, Expressing, Plasmid Preparation, Binding Assay, Sequencing

Journal: Methods in Molecular Biology

Article Title: Monoclonal Antibodies

doi: 10.1007/978-1-62703-992-5

Figure Lengend Snippet: Fig. 2 Examples of gels exemplifying the different stages of the cloning process. ( a ) Heavy ( 1 , 3 ) and light ( 2 , 4 ) chain RT-PCR products, linker fragment ( 5 ), fusion PCR products ( 6 , 7 ), and linearized expression plasmid ( 8 ). ( b ) Examples for colony PCR products: recombinant antibodies with specifi c light chain ( 1 , 3 ) or myeloma aber- rant light chain ( 2 , 4 ), negative control without insert ( 5 ), positive control ( 6 ), myeloma control ( 7 ). Primer 45 ( see Table 1 ) hybridizes in the plasmid (3′ end of the heavy constant region) and primer 46 within the linker fragment (light constant 3′ UTR + CMV promoter for heavy chain) resulting in a PCR product of 2.4 kb. In the presence of the myeloma aberrant chain primer 47 hybridizes as well resulting in an additional 3 kb fragment. In clones without any insert, a 2.1 kb product is produced due to hybridizing of primer 46 to the CMV promoter region present in the plasmid

Article Snippet: Amplify the linker fragment from one of our recombinant antibodies (e.g., SI3-Flrt3, Addgene ID: 28217; http://www. addgene.com ).

Techniques: Cloning, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Recombinant, Negative Control, Positive Control, Control, Clone Assay, Produced

Journal: Scientific Reports

Article Title: Obinutuzumab induces lysosomal destabilization via sphingomyelin-dependent inhibition of TRPML2

doi: 10.1038/s41598-026-38087-5

Figure Lengend Snippet: Lysosomal stress and TRPML inhibition enhance obinutuzumab-mediated direct cell death. ( a ) LMP and DCD were measured in Raji cells treated with hypertonic medium (200 mM sucrose) or hypotonic medium (60% distilled water in complete culture medium) for 2 h in the presence of OBI (Hereafter, the concentration of OBI is 10 μg/ml unless otherwise stated). LMP and DCD were evaluated by LysoTracker Green and propidium iodide (PI) staining. One-way ANOVA with Dunnett’s multiple-comparisons test (vs. control). ( b ) OBI-induced LMP and DCD were assessed in Raji cells co-incubated with increasing concentrations of the TRPML inhibitor (1R,2R)-ML-SI3 (10 µM). In parallel, cells pretreated overnight with the PIKfyve inhibitor Apilimod (50 nM) showed enhanced OBI responses, but this effect was abolished when TRPMLs were simultaneously inhibited. One-way ANOVA with Dunnett’s multiple-comparisons test (vs. control). ( c ) Dose-dependent enhancement of OBI-induced DCD was observed following 2-h co-incubation with a fixed concentration of the (1R,2R)-ML-SI3 (10 µM, over 2 h incubation, this (1R,2R)-ML-SI3 treatment condition was used unless otherwise stated). ( d , e ) Time-dependent enhancement of OBI-induced LMP ( d ) and DCD ( e ) were assessed in TRPMLs-targeting siRNA cell lines. ( f ) Lysosome enlargement and fluorescence intensity were indicated by LT Deep Red staining, then imaged by confocal microscopy. (1R, 2R)-ML-SI3, hypertonic, and hypotonic medium were incubated 2 h. Scale bars in all images represent 5 µM. ( g , h ) Quantification of cathepsin B release induced by OBI and (1R,2R)-ML-SI3 treatment. ( g ) Raji cells were treated with (1R,2R)-ML-SI3, hypertonic medium (200 mM sucrose), or hypotonic medium (60% distilled water in complete medium) for 2 h, and with OBI for 4 h. Cathepsin B releases were visualized by confocal microscopy by immunostaining. ( h ) The summary of the release data shows significant cathepsin B secretion in response to OBI treatment, which was further modulated by (1R,2R)-ML-SI3 and osmotic stress conditions. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001 compared to obi-treated group.

Article Snippet: For drug treatments, (1R,2R)-ML-SI3 (HY-134819A; MCE), ML2-SA1 (axon Medchem), and 3- O -methyl Estradiol (#42995, Cayman Chemical) were administered for 2 h at 37 °C, followed by OBI (0.3 μg/ml) treatment for an additional 2 h. Endocytosis inhibition was performed using filipin (HY-N6716, MCE), dynasore (S8047, Sellekchem), pitstop2 (HY-115605, MCE), Sphingomyelinase (S7651-10UN, Sigma), or heat-inactivated Sphingomyelinase for 30 min at 37 °C before antibodies treatment (10 μg/ml, IgG, RTX, OBI).

Techniques: Inhibition, Concentration Assay, Staining, Control, Incubation, Fluorescence, Confocal Microscopy, Immunostaining