plenti sgrb1 cre vector  (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: 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 , Position and sequence of the PYD-FISNA linker in human and zebrafish NLRP3, and zebrafish NLRP3 containing human PYD-FISNA linker sequence encoded by the exon 3 of human NLRP3 gene (red dashed box). Sequences corresponding to FISNA domain and polybasic region are indicated with black lines. Cys130 palmitoylated in human NLRP3 is highlighted in blue. b , SDS-PAGE gels of sucrose gradient fractions of zebrafish NLRP3 WT and zebrafish NLRP3 containing human linker sequence. c , Sequence alignment of zebrafish and human NLRP3. Domains are indicated with lines and color-coded. Residues forming a “face-to-face” interface according to the human (PDB: 7PZC) and mouse (PDB: 7LFH) NLRP3 “cage” structures are indicated with red and orange stars, respectively. Residues forming a “back-to-back” interface according to the human (PDB: 7PZC) and mouse (PDB: 7LFH) NLRP3 “cage” structures are indicated with blue and violet stars, respectively. Numbers correspond to alignment positions within the full-length sequence alignment. d , Position and sequence of known palmitoylation sites in human and zebrafish NLRP3. Palmitoylated residues are highlighted in blue.

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

Techniques: Sequencing, SDS Page