mouse calnexin primary  (Proteintech)

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet: (A) SAR1B nitrosylates SURF4. FLAG-tagged SNO-SAR1B was incubated with V5-tagged SURF4. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. (B) SURF4 nitrosylates PCSK9. V5-tagged SNO-SURF4 was incubated with FLAG-tagged PCSK9. Reaction mixtures were subjected to SNO-RAC and SNO-proteins visualized by western blot. Representative image (n = 3) is shown. Mature PCSK9 is visualized in SNO-PCSK9 lanes. (C) Western blot analysis of SNO-PCSK9 and SNO-SAR1B in SCoR2-deficient HEK293 transfected with wild-type PCSK9 and wild-type SAR1B and treated with 200 μM ethyl ester S-nitroso-cysteine (ECySNO) for 90 min. Anti-FLAG antibody was used to visualize SAR1B. (D) Quantification (n = 3) of SNO-PCSK9 and SNO-SAR1B (normalized to total PCSK9 and SAR1B, respectively) from (C) and related experiments. (E) Western blot analysis of SNO-SAR1B wild-type and indicated mutations in SCoR2-deficient HEK293 transfected with SAR1B wild-type and indicated mutations and treated with 200 μM ECySNO for 90 min. Anti-FLAG antibody was used to visualize SAR1B in a single experiment that is verified in subsequent assays. (F) Western blot analysis of SNO-PCSK9, SNO-SURF4, and SNO-SAR1B in SCoR2-deficient HEK293 cells transiently overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (G) Quantification (n = 3) of SNO-PCSK9 (mature band, normalized to total mature PCSK9) and SNO-SURF4 from (F). (H) Representative western blot analysis for cellular and secreted (media) PCSK9 in SCoR2-deficient HEK293 cells overexpressing SAR1B WT or SAR1B C102A/C178A and treated with 200 μM ECySNO for 90 min prior to harvest. (I) Quantification (n = 3) of secreted (media) PCSK9 (normalized to mature PCSK9 band) from (H). p values in (I) were calculated by one-way ANOVA. (J) SCoR-deficient HEK293 cells stably expressing PCSK9 were treated with or without 200 μM ECySNO (+SNO) for 90 min then stained with anti-PCSK9 (green) and anti-calnexin (red, ER marker) antibodies. Scale bar, 5 μm. (K) Quantification of mean PCSK9 signal intensity in pixels positive for calnexin (n = 12 cells per condition). Control: SNO-RAC assay performed without ascorbate. See also .

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Incubation, Western Blot, Transfection, Stable Transfection, Expressing, Staining, Marker, Control

Journal: Cell reports

Article Title: A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis

doi: 10.1016/j.celrep.2022.111538

Figure Lengend Snippet:

Article Snippet: For PCSK9 visualization experiments, cells were stained with rabbit PCSK9 primary (Proteintech) and mouse Calnexin primary then stained with Alexa488-conjugated anti-rabbit and Alexa594-conjugated anti-mouse secondary antibodies.

Techniques: Recombinant, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, shRNA, Mutagenesis, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Regulatory T Cell Extracellular Vesicles Modify T-Effector Cell Cytokine Production and Protect Against Human Skin Allograft Damage

doi: 10.3389/fcell.2020.00317

Figure Lengend Snippet: Activated human Tregs release EVs. (A) (1st panel) Representative EM image of EVs released by activated human Treg cells. Scale bar indicates 100 nm. 2nd panel, A representative of a size distribution plot for EVs released by TCR activated Tregs. The EV samples were acquired using the NanoSight LM-10 and 5 videos of 30 s duration were recorded. Histogram represents the mean size from all 5 measurements with the red error bars indicating mean ± 1 SEM. 3rd panel, the average mean particle size and the average mode size of Treg EVs isolated from 12 individual donors. (B) Graph shows the number of EVs released per cell from resting versus activated Tregs. EVs were isolated from Tregs expanded from 5 donors. (C) Representative flow cytometry histogram plots showing the expression of CD63, CD81, CD25, CTLA4, CD39, CD73, Fas-Ligand, CD4, and CCR4 on Treg EVs attached to latex beads following specific antibody staining (black lines). Control samples (gray lines) represent beads stained with the aforementioned antibodies in the absence of EVs. Values indicate the mean fluorescence intensity (MFI) for each of the molecules shown and the control beads. Data represents 1 out of 3 independent experiments. (D) Left panel, graph showing the average number of CD63 + EVs (×10 9 ) ± SEM detected from 1 × 10 6 Tregs isolated from 3 donors using a CD63 ExoELISA (left panel). Right panel, Western blot showing CD63 (top panel), CD81 (middle panel) and Calnexin (lower panel) expression in Treg EVs lysates (2 individual donors) and Treg cell lysates (1 individual donor). (E) Normalized Total Spectra of described proteins found in Treg EVs (1 individual donor).

Article Snippet: Calnexin expression was assessed using a rabbit anti-human/mouse Calnexin primary Ab (Proteintech, Manchester, United Kingdom) followed by a goat anti-rabbit Ig-HRP conjugated secondary Ab (Cell Signaling Technologies MA, United States), both at a 1:1000 dilution.

Techniques: Isolation, Flow Cytometry, Expressing, Staining, Control, Fluorescence, Western Blot