cd63 pegfp c2  (Addgene inc)

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) WT and Peli1-KO BMDMs were treated with 1 µg/mL LPS for 3.5 h with (+) or without (−) further stimulation with ATP, nigericin, alum, poly(dA:dT), flagellin, and MDP. The conditioned media were collected and subjected to ELISA to determine the level of IL-1β and IL-18 (WT, n = 3; KO, n = 3). (B) WT and Peli1-KO BMDMs were either NT or primed with 1 µg/mL LPS for 3.5 h followed by a further incubation without (NT) or with the indicated NLRP3 inducers. Cell lysates and precipitated conditioned media (Sup) were analyzed by immunoblotting to detect the indicated proteins. (C) WT and Peli1-KO BMDMs were NT (−) or primed with 500 ng/mL Pam3CSK4 for 3.5 h with (+) or without (−) further stimulation with ATP and nigericin for 30 min. Conditioned media were collected for ELISA to determine the level of IL-1β and TNF-α (for IL-1β: WT, n = 3; KO, n = 3; for TNF-α: WT, n = 4; KO, n = 4). (D) WT and Peli1 KO BMDMs were primed with (+) 500 ng/mL Pam3CSK4 for 3.5 h and then further incubated without (NT) or with the indicated inflammasome inducers. Cell lysates and precipitated conditioned media (Sup) were analyzed by immunoblotting to detect the indicated proteins. (E and F) 8-week-old Peli1-KO or WT control mice were i.p. injected with MCC950 for 2 h before LPS challenge. The serum was collected 2 h later for IL-1β ELISA, and mouse viability was monitored daily (WT, n = 9; KO, n = 10). Data are representative of three independent experiments, and bar graphs are presented as mean ± SEM of biological replicates. p values were determined by a two-tailed unpaired Student’s t test (A, C and F) or Kaplan-Meier method with a log-rank test (E): *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) Immunoblot analysis of ASC in whole-cell lysates (upper) and ELISA of IL-1β in conditioned media (lower) of control and ASC-knockdown iBMDMs treated as indicated (WT, n = 4; KO, n = 4). (B) Immunoblot analysis to detect ubiquitination of ASC immunoprecipitated from lysates of ASC-knockdown iBMDMs stably transduced with (+) ASC WT or K55R and primed with LPS for 3 h with further stimulation for 1 h with nigericin. (C and D) ASC-knockdown iBMDMs reconstituted (by retroviral infection) with (+) or without (−) ASC WT or K55R were primed with LPS for 3 h and further stimulated for 1 h with nigericin. Inflammasome activation was determined by immunoblotting analysis of precipitated conditional media (Sup) or cell lysates (C) or ELISA analysis of IL-1β in conditioned media (D) (WT, n = 3; KO, n = 3). (E) CoIP analysis to determine the interaction of endogenous NLRP3 with ASC or ASC K55R using ASC-knockdown iBMDMs rescued with (+) or without (−) ASC WT or K55R. The cells were primed with LPS for 3 h and further stimulated for 1 h with nigericin. (F) CoIP assays to determine Peli1-stimulated interaction between transfected NLRP3 and ASC or ASC K55R in 293T cells. (G) ASC-knockdown iBMDMs rescued with (+) or without (−) ASC WT or K55R were primed with LPS for 3 h and further stimulated for 1 h with nigericin. ASC oligomerization was detected by anti-Myc immunoblot using insoluble cross-linked fraction (upper), and ASC expression was analyzed using soluble fraction. Data are representative of three independent experiments, and bar graphs are presented as mean ± SEM. p values were determined by a two-tailed unpaired Student’s t test (A and D): **p < 0.01; ***p < 0.001.

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Stable Transfection, Transduction, Retroviral, Infection, Activation Assay, Transfection, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-Flag-Caspase-1 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75128).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-Flag-ASC , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75134).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-HA-NEK7 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75142).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Immunoblotting analysis of STX1A knockdown and control cells for checking the expression of STX1A. * non-specific band highlighted by anti-STX1A antibody. The fold change in band intensities was normalized with internal control and indicated on the blots. (B) Immunoblotting analysis of GFP-STX1A overexpression in HeLa cells. GFP expression is used as a control. The blot was probed with an anti-GFP antibody. γ-tubulin is used as the loading control in all immunoblots. (C) Images represent the GFP-STX1A overexpression in HeLa cells. Undeconvolved (left) and deconvolved (right) images are shown separately. Insets are magnified views of the white boxed areas. (D-F) Live cell imaging microscopy of HeLa cells expressing GFP-STX1A and LAMP1-RFP in D; mCherry-STX1A and GFP-CD63 in E; and GFP-CD63 and LAMP1-RFP in F. Insets are magnified views of the white boxed areas shown in the images at different time points (s, Sec). Scale bars, 10 µm. (G) Subcellular membrane fractionation of HeLa cells. Fractions were probed for STX1A, LAMP1 (lysosome marker), Na + -K + ATPase (plasma membrane marker) and Rab22A (recycling endosome marker). In- Input and numbers represent the membrane (alternative) fractions from top to bottom of the sucrose gradient. (H) Plot represents the number of LysoTracker puncta in non-transfected (-) and GFP-STX1A overexpressing cells. (I) Plot represents the CTCF (A.U.) of DQ-Red intensity in non-transfected (-) and GFP-STX1A overexpressing cells. (J) Plot represents the size of lysosomes (μm 2 ) in non-transfected (-) and GFP-STX1A overexpressing cells. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant and *p≤0.1.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Western Blot, Knockdown, Control, Expressing, Over Expression, Live Cell Imaging, Microscopy, Membrane, Fractionation, Marker, Transfection

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, E) IFM analysis of HeLa cells transfected with control or two different STX1A (1 and 2) siRNA sequences. The cells were stained for LAMP1 and LBPA or CD63 (A). Another set of cells was overexpressed with RFP-LC3 or tfLC3 (rat LC3 fused to mRFP and EGFP, E). RFP-LC3 expressing cells were stained for LAMP1. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) between LAMP1 and LBPA, shown in A. (C, D, G) Immunoblotting analysis of control and STX1A knockdown cell lysates. The blots were probed for checking the expression of different lysosome associated proteins in C, cargo proteins in D, and autophagy related proteins in G. γ- tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (F) Plots represent the number of RFP-LC3 puncta, Pearson’s correlation coefficient ( r value) between LAMP1 and RFP-LC3, and the number of autolysosomes. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant, **p≤0.01, and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transfection, Control, Staining, Expressing, Western Blot, Knockdown