expression vectors pnic yfp  (Addgene inc)

Journal: bioRxiv

Article Title: Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

doi: 10.64898/2026.04.17.719140

Figure Lengend Snippet: a , LDH release assay in NLRP3-KO BlaER1 cells reconstituted with the indicated FLAG-tagged NLRP3 constructs or control plasmid. Expression was induced with doxycycline (Dox) for 18 h. Cells were primed with 200 ng/ml LPS for 4h followed by activation with 6.5 µM nigericin, 30 µg/ml imiquimod or 0.025 mg/ml needle toxin for 2 h. Data are represented as mean ± SD, n = 3. b , Western blots of the whole cell lysates from THP-1 NLRP3-KO cells reconstituted with human or zebrafish NLRP3 or zebrafish NLRP3 containing human PYD. Cells were treated with 1 µg/ml LPS for 3 h followed by activation with 20 µM nigericin or 200 µM imiquimod for 1 h. FLAG-tagged NLRP3 constructs and cleaved GSDMD were visualized with corresponding antibodies. c, d, ASC speck formation in HEK293T cells stably expressing YFP-ASC, upon transfection with indicated NLRP3 constructs: human, zebrafish NLRP3 and zebrafish NLRP3 with B30.2-domain deletion (zebrafish NLRP3 ΔB30.2 ) ( c ); wild-type and oligomer-disrupting mutants of zebrafish NLRP3 ( d ). Cells were transfected with the indicated amount of DNA, and the number of ASC specks per image area was calculated 24 h post-transfection. Data are represented as mean ± SD, n = 3.

Article Snippet: Human full-length ASC was cloned into pLenti-EF1a-C-tGFP (Origene, PS100072) and tGFP was exchanged with YFP.

Techniques: Lactate Dehydrogenase Assay, Construct, Control, Plasmid Preparation, Expressing, Activation Assay, Western Blot, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

doi: 10.64898/2026.04.17.719140

Figure Lengend Snippet: a , Western blot analysis of NLRP3 expression in reconstituted cell lines in following doxycycline (Dox) induction. Bands corresponding to FLAG-tagged NLRP3 constructs are indicated with arrows. b, c , Western blot analysis of the whole cell lysates from HEK293T cells reconstituted with YFP-ASC and transfected with the indicated NLRP3 constructs, demonstrating relative expression levels of NLRP3 constructs used for ( b ) and ( c ). FLAG-tagged NLRP3 constructs and β-actin were visualized with corresponding antibodies. Cells were transfected with 100 ng DNA per well in a 96-well plate, and the lysates were collected 24 h post-transfection.

Article Snippet: Human full-length ASC was cloned into pLenti-EF1a-C-tGFP (Origene, PS100072) and tGFP was exchanged with YFP.

Techniques: Western Blot, Expressing, Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Mapping the MOB proteins’ proximity network reveals a unique interaction between human MOB3C and the RNase P complex

doi: 10.1016/j.jbc.2023.105123

Figure Lengend Snippet: MOB3C interacts with catalytically active RNase P complex. A , coimmunoprecipitation assay for YFP-tagged POP1 and RPP30 with FLAG-tagged MOB3C in HeLa cells. B , schematic outline for the affinity purification–mass spectrometry (AP–MS) with DSP crosslinking (CL). A Western blot analysis validating the used cell lines in AP–MS is shown. C , Venn diagram showing the commonly defined proteins in the BioID and crosslinked AP–MS datasets. The table displays the average spectral counts of each RNase P subunit defined in the AP–MS dataset. D , a proximity network of the RNase P protein subunits defined in both the MOB3C BioID and AP–MS datasets in reference to BioGrid (see the section). E , pre-tRNA cleavage assay of the pull-down experiments using GST-MOB1A or GST-MOB3C. Time-course reactions showing RNase P activity when GST-MOB3C, but not GST-MOB1A, was used. +: a positive control of pre-tRNA Arg cleaved by recombinant Escheichia coli RNase P; –: a negative control of pre-tRNA Arg without enzyme; I: input. F , SDS-PAGE analysis confirms the presence of GST-MOB3C and GST-MOB1A post-pulldown. DSP, dithiobis (succinimidyl propionate); EV, empty vector; GST, glutathione- S -transferase; MOB, monopolar spindle-one-binder.

Article Snippet: YFP-POP1 expression vector was generated by amplifying the POP1 cDNA from pCMVh-POP1-3xFLAG (gift from Martin Dorf; Addgene #53968) with flanking XhoI and SalI restriction sites and ligating it into the multicloning site of YFP-C1 backbone vector by swabbing out the YFP-RPP30.

Techniques: Co-Immunoprecipitation Assay, Affinity Purification, Mass Spectrometry, Western Blot, Cleavage Assay, Activity Assay, Positive Control, Recombinant, Negative Control, SDS Page, Plasmid Preparation