card12 antibody  (Bio-Techne corporation)

Journal: International Journal of Molecular Sciences

Article Title: Comparison of Pollutant Effects on Cutaneous Inflammasomes Activation

doi: 10.3390/ijms242316674

Figure Lengend Snippet: mRNA expression levels of NLRP3 ( a ), NLRP1 ( b ), NLRC4 ( c ) and NLRP6 ( d ) in human skin biopsies exposed to air pollutants and collected at DAY 1, DAY 2 and DAY 4 post exposure. ( e ) Protein expression levels of ASC in human skin biopsies exposed to air pollutants and analyzed at the indicated timepoints. The protein expression level was quantified using ImageJ software 1.53a (Java 1.8.0_172) and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001 pollutants vs. Ctrl at the respective timepoints (DAY 1 and DAY 4) by 2-way ANOVA followed by Tukey’s post-hoc comparison test.

Article Snippet: Sections were then incubated overnight with primary antibodies at 4 °C as follows: NLRP1 (sc-166368Santa Cruz Biotechnology Inc., Dallas, TX, USA), NLRP3 (cat. NBP1-97601, Novus Biological, Littleton, CO, USA) 1:100, NLRP6 (NBP2-31372, Novus Biological, Littleton, CO, USA) 1:200, NLRC4 (NB100-561425, Novus Biological, Littleton, CO, USA) 1:200, ASC (cat. NBP1-78977, Novus Biological, Littleton, CO, USA) 1:150 or ASC (sc-271054, Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 8-OHdG (sc-393871, Santa Cruz Biotechnology Inc., Dallas, TX, USA), type I collagen (ab-138492, abcam, Cambridge, UK), Sestrin 2 (cat. 10795-1-AP, Proteintech, Rosemont, IL, USA), 4HNE (AB5605, MilliporeSigma, Burlington, MA, USA) PBS, BSA 0.25%.

Techniques: Expressing, Software, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Comparison of Pollutant Effects on Cutaneous Inflammasomes Activation

doi: 10.3390/ijms242316674

Figure Lengend Snippet: Immunofluorescence staining for NLRP3 (green staining) and ASC (red staining) ( a ); NLRP1 (red staining) and ASC (green staining) ( b ); NLRC4 (green staining) and ASC (red staining) ( c ); and NLRP6 (green staining) and ASC (red staining) ( d ) in human skin biopsies exposed to air pollutants and collected at the indicated timepoints. Blue staining (DAPI) represents nuclei. Images were taken at 40× magnification and the fluorescent signal was quantified using the ImageJ software 1.53a (Java 1.8.0_172). The colocalization between NLRP3, NLRP1, NLRC4, or NLRP6 and ASC is expressed as Pearson’s correlation coefficient. Data are the results of the averages of at least three different experiments, * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001 pollutants vs. Ctrl at the respective timepoints (DAY 1 and DAY 4) by 2-way ANOVA followed by Tukey’s post-hoc comparison test.

Article Snippet: Sections were then incubated overnight with primary antibodies at 4 °C as follows: NLRP1 (sc-166368Santa Cruz Biotechnology Inc., Dallas, TX, USA), NLRP3 (cat. NBP1-97601, Novus Biological, Littleton, CO, USA) 1:100, NLRP6 (NBP2-31372, Novus Biological, Littleton, CO, USA) 1:200, NLRC4 (NB100-561425, Novus Biological, Littleton, CO, USA) 1:200, ASC (cat. NBP1-78977, Novus Biological, Littleton, CO, USA) 1:150 or ASC (sc-271054, Santa Cruz Biotechnology Inc., Dallas, TX, USA) 1:50, 8-OHdG (sc-393871, Santa Cruz Biotechnology Inc., Dallas, TX, USA), type I collagen (ab-138492, abcam, Cambridge, UK), Sestrin 2 (cat. 10795-1-AP, Proteintech, Rosemont, IL, USA), 4HNE (AB5605, MilliporeSigma, Burlington, MA, USA) PBS, BSA 0.25%.

Techniques: Immunofluorescence, Staining, Software, Comparison

Journal: Virology

Article Title: SARS-CoV-2 viroporin encoded by ORF3a triggers the NLRP3 inflammatory pathway

doi: 10.1016/j.virol.2022.01.003

Figure Lengend Snippet: ORF3a activates the NEK7-NLRP3 inflammasome via ASC-dependent and independent modes. (A) Lysates of FLAG-ORF3a- or empty vector (EV)-transfected HEK-293T (left) or A549 (right) cells were immunoblotted with indicated antibodies. In parallel, A549 cells transfected with FLAG-ORF3a or EV were examined for NLRP3 transcripts using RT-PCR (panel beneath A549 immunoblot). (B, D) HEK-293T (left) or A549 (right) cells were co-transfected with FLAG- ORF3a and control siRNA (B, D), NLRP3 siRNA (B), or NEK7 siRNA (D) for 24 h prior to immunoblotting with indicated antibodies. (C) HEK-293T (left) or A549 (right) cells were transfected with EV or FLAG-ORF3a and exposed to MCC950 for 24 h prior to immunoblotting. (E) Lysates of cells transfected with ORF3a or EV were immunoblotted with indicated antibodies. (F, G) A549 cells were co-transfected with FLAG-ORF3a and control siRNA, ASC siRNA (F), NLRP1 siRNA (G; left), or NLRC4 siRNA (G, right) for 24 h prior to immunoblotting with indicated antibodies. Experiments were performed at least thrice.

Article Snippet: The following antibodies were used: rabbit anti-Caspase-1 antibody (Thermo Scientific, Cat. PA587536), rabbit anti-cleaved Caspase-1 antibody (Thermo Scientific, Cat. PA538099), mouse anti-IL-1β (Cell Signaling Technology, Cat. 12242s), mouse anti-Flag M2 antibody (Sigma-Aldrich, Cat. F1804), mouse anti-β-actin antibody (Sigma-Aldrich, Cat. A5441), rabbit anti-phospho-IκBα (Ser32) antibody (Cell Signaling Technology, Cat. 2859s), rabbit anti-IκBα antibody (Cell Signaling Technology, Cat. 9242s), rabbit anti–NF–κB p65 antibody (Cell Signaling Technology, Cat. 8242s), rabbit anti-Caspase 3 antibody (GeneTex, Cat. GTX110543), rabbit anti-Gasdermin D (L60) antibody (Cell Signaling Technology, Cat. 93709s), rabbit anti-cleaved-Gasdermin D (Asp275) antibody (Cell Signaling Technology, Cat. 36425s), rabbit anti-NLRP3 antibody (Invitrogen, Cat. PA5-21745), rabbit anti-NEK7 antibody (Cell Signaling Technology, Cat. 3057s), rabbit anti-ASC antibody (Cell Signaling Technology, Cat. 13833s), rabbit anti-NLRP1 antibody (Novus Biologicals, Cat. NB100–56147SS), rabbit anti-NLRC4 antibody (Novus Biologicals, Cat. NB100–56142SS), rabbit anti-SARS-CoV-2 ORF3a antibody (FabGennix, Cat. SARS-COV2-ORF3A-101AP), HRP-conjugated goat anti-mouse IgG(H + L) (Thermo Scientific, Cat. 626520) and HRP conjugated goat anti-rabbit IgG(H + L) (Thermo Scientific, Cat. 31460), and HRP conjugated goat anti-rabbit IgG (light chain) (Novus, Cat. NBP2-75935).

Techniques: Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control