Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: Formula determining rates where new podocytes derived from CoRL

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Derivative Assay, Migration

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: Renin + CoRL and RFP + CoRL in JGC

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques:

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: RAAS inhibition increases the number of labeled cells of renin lineage in the juxtaglomerular compartment in non-diseased mice renin/red fluorescent protein co-staining. Confocal microscopy showing co-staining for renin (green color, first column) and the RFP reporter (red color, middle column) as single panels. A merge of the two panels creates a yellow color if renin and RFP staining co-localize (right column). Nuclei stain blue with DAPI. The quantitative data is shown in panel D. (A) Non-diseased mice given water: When healthy, non diseased Ren1cCreERxRs-tdTomato-R mice were given tamoxifen, and given water alone to drink for 25 days, renin (A, i) and RFP staining (A, ii) were restricted to co-localized in the juxta-glomerular compartment (JGC) (A, iii). (A, iv – A,vi) Higher magnification images of the insets shown in the figures above. (B) Non-diseased mice given enalapril: Administering the ACE inhibitor enalapril in the drinking water for 25 days to healthy, non-diseased Ren1cCreERxRs-tdTomato-R CoRL reporter mice increased the number of cells in the JGC staining for renin (B, i) and RFP (B, ii). Renin and RFP staining merged, and was restricted to and co-localized in the JGC (B, iii). Higher magnification images of the JGC insets are shown for renin staining (B, iv), RFP staining (B, v) and merged (B, vi). (C) Non-diseased mice given losartan: non-diseased Ren1cCreERxRs-tdTomato-R CoRL reporter mice treated with ARB losartan in drinking water for 25 days increased the number of cells in the JGC staining for renin (C, i) and RFP (C, ii). The majority of cells merged. Higher magnification images of the JGC are shown for renin staining (C, iv), RFP staining (C, v) and merged (C, vi). (D) Quantitative Data: Quantification of the percentage of all cells in the kidney cortex expressing RFP showed a significant increase in healthy, non-diseased animals given either enalapril or losartan compared to water alone control mice. Original magnification is ×400. Data presented as the mean±SD. These results show that in non-diseased reporter mice, RAAS inhibition increases the number of renin stained cells, and labeled cells of renin lineage and significantly increased the percentage of cells in the total kidney cortex cell population that comprised RFP+CoRL compared to mice given water alone. Data are represented as mean ±SD.

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Staining, Confocal Microscopy, Expressing, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: RAAS inhibition increases the number of labeled cells of renin lineage in the juxtaglomerular compartment and migration of fate-mapped cells of renin lineage in FSGS mice (A–C) Shows confocal images of co-staining for renin (green, first column) and the RFP reporter (red, middle column), and when the images were merged (right column); (D, E) shows quantitative data. Following the induction of experimental FSGS in Ren1cCreERxRs-tdTomato-R mice, diseased mice were randomized on day 3 to receive water, Hydralazine, Enalapril or Losartan in their drinking water for 25 days. Mice were analyzed at FSGS day 28. (A) Hydralazine treated FSGS mice: (A, i) In FSGS mice given Hydralazine, staining for renin was restricted to the juxta-glomerular compartment (JGC). (A, ii). RFP staining was detected in both the JGC and the intra-glomerular compartment (IGC, arrow shows example). (A, iii). RFP staining co-localized with renin staining (yellow) in the JGC, but not in the IGC. (A, iv–vi). Higher magnification images of the JGC are shown for renin staining (A, iv), RFP staining (A, v) and merged images (A, vi). (B) Enalapril treated FSGS mice: The ACE inhibitor Enalapril was added to the drinking water on day 3 FSGS for 25 days. (B, i) Renin was restricted to the JGC (B, ii). RFP staining increased in the JGC, and was readily detected in glomeruli in the IGC (arrows). (B, iii). RFP staining co-localized with RFP in the JGC. Higher magnification images of the insets for renin staining (B, iv), RFP staining (B, v) and merged images (B, vi) showing that renin localizes to the JGC, and that RFP+CoRL that have migrated to the IGC lack renin staining. (C) Losartan treated FSGS mice: Double staining for renin and RFP in FSGS mice given Losartan showed staining for renin in the JGC (C, i). RFP staining was present in the JGC and in the IGC (arrows show examples) (C, ii). RFP staining co-localized with renin in the JGC (yellow), but did not co-localize in the IGC (C, iii, arrows). Higher magnification images for renin staining (A, iv), RFP staining (A, v) and merged images (A, vi) show that RFP+CoRL in the IGC do not co-express renin. (D, E) Quantification of RFP+ cells in the juxta- (JGC) and intra- (IGC) glomerular compartment: (D) The percentage (%) of RFP+CoRL shown on the Y-axis was derived by dividing the total number of RFP stained cells in the JGC by the total number of DAPI positive cells in the kidney cortex. Enalapril and Losartan increased the % of RFP+CoRL in the JGC. (E) The percentage (%) of RFP+CoRL shown on the Y-axis was derived from the ratio of RFP+CoRL in the intra-glomerular compartment (IGC) that derived from the juxta-glomerular compartment (JGC). There was a trend in the Enalapril group to increased migration of RFP+CoRL from the JGC to the IGC in FSGS. Data presented as the mean ± standard deviation.

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Migration, Staining, Double Staining, Derivative Assay, Standard Deviation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: RAAS inhibition increases the number of labeled CoRL in the JGC and migration of fate-mapped CoRL in FSGS mice. (A–C) Confocal microscopy of co-staining of BrdU (green, left column), red fluorescent protein (RFP, red, middle column) and DAPI (blue), and merged images (yellow, right column) (A) Enalapril in non-diseased mice: (A, i) BrdU stained cell detected in the JGC; (A, ii) RFP stained cells in the JGC; (A, iii) BrdU stained cell co-localizes with RFP+ cell in the JGC (arrow head). (A, iv–vi) high power magnification of insets shown in figures above (B) Losartan in non-diseased mice: (B, i) BrdU stained cells detected in the JGC; (B,ii) RFP stained cells representing labeled cells of renin lineage; (B, iii) several RFP+ cells co-stain with BrdU (arrow heads). (B, iv–vi) high power magnification of insets shown in figures above (C) Enalapril in FSGS mice: (C, i) BrdU staining was detected in cells in the JGC (inset) and also in other cells in the tubule-interstitium; (C, ii) the number of RFP stained cells are higher in the JGC; (C, iii) BrdU co-stains in a subset RFP+CoRL in the JGC (arrow heads), but not RFP+CoRL in the IGC. (C, iv–vi) High power magnification of insets shown in figures above. (D) Losartan in FSGS mice: (D, i) BrdU staining was detected in cells in the JGC (inset); (D, ii) an increase in the number of RFP stained cells in the JGC (inset); (D, iii) BrdU co-staining in RFP+CoRL in the JGC (arrow head), but not RFP+CoRL in the IGC (Arrows). (D, iv–vi) high power magnification of insets shown in figures above.

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Labeling, Migration, Confocal Microscopy, Staining, BrdU Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: RFP + CoRL that migrated to the IGC

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Labeling

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: CoRL express podocyte proteins and show ultrastructural features of podocytes in CoRL reporter mice with experimental FSGS after RAAS inhibition. (A) Confocal microscopy of double-staining for synaptopodin (synpo, green) and RFP (red). (A, i) in FSGS mouse at day 28, Synpo staining is restricted to podocytes; (A, ii) RFP staining detected within glomerulus (inset); (A, iii) Synpo and RFP merge (yellow, arrowhead) in the glomerulus. (A, iv–vi) Higher-power magnification of insets shown above. Note the morphology of the RFP+CoRL in the glomerulus in (A, v) that resembles characteristic podocyte features. (B) Confocal microscopy of WT-1 (green) and RFP (red) staining. (B, i) WT-1 staining is detected in the glomerulus in the nucleus in a typical podocyte distribution, and an occasional WT-1–stained cell is detected in the cytoplasm in the JGC. (B, ii) RFP staining is detected in the JGC and IGC. (B, iii) The merge shows that cells in the inset costain for nuclear WT-1 and for RFP. (B, iv–vi) High power of the insets above. Arrowhead shows example of cell in the glomerulus costaining for nuclear WT-1 and RFP. (C–E) Transmission electron micrograph of RFP-stained cells: (C) Renin granules are noted (black arrows). Arrowheads show RFP-labeled CoRL. (D) RFP does not colocalize with podocytes. (E) Subset of RFP stained cells have foot processes (arrowheads) characteristic of podocytes.

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Confocal Microscopy, Double Staining, Staining, Transmission Assay, Labeling

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: Transdifferentiation of RFP + CoRL to different glomerular cell types in the IGC

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques:

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Renin-Angiotensin-Aldosterone System Inhibition Increases Podocyte Derivation from Cells of Renin Lineage

doi: 10.1681/ASN.2015080877

Figure Lengend Snippet: CoRL express PEC and mesangial cell proteins in CoRL reporter mice with experimental FSGS after RAAS inhibition. (A) Confocal microscopy of PAX8 (green) and RFP (red) costaining. (A, i) PAX8 staining detected in classic PEC distribution along Bowman’s capsule. (A, ii) RFP-stained cell along Bowman’s capsule within inset box. (A, iii) Cell along Bowman’s capsule costaining for RFP and PAX8. (A, iv–vi) Higher-power magnification of insets above. (B) Confocal microscopy of (B, i) α8 integrin staining detected in a classic mesangial cell distribution. (B, ii) RFP-stained cells in the JGC and in the glomerulus. (B, iii) Cell costaining for α8 integrin and RFP. (B, iv–vi) Higher-power magnification of insets above.

Article Snippet: Immunoelectron Microscopy To better visualize the morphology of CoRL when in a glomerular location, immunoperoxidase staining was performed with a rabbit antibody to RFP (Rockland Immunochemicals for Research).

Techniques: Inhibition, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: Enrichment of GABARAP Relative to LC3 in the Axonal Initial Segments of Neurons

doi: 10.1371/journal.pone.0063568

Figure Lengend Snippet: (A) Expression of GFP-GABARAP in tissues of GFP-GABARAP transgenic mice. Cell lysates were immunoblotted with anti-GFP antibody to detect GFP-GABARAP. Endogenous GABARAP and LC3 were detected with anti-GABARAP and anti-LC3 antibodies, respectively. Arrow-heads indicate GFP-GABARAP; asterisks indicate non-specific bands in the skeletal muscle. CON, lysate from control mouse tissue; tg, lysate from GFP-GABARAP transgenic mouse tissue; SK muscle, skeletal muscle. ( B – I ) Confocal fluorescence images of GFP-GABARAP in the heart ( B and C ), liver ( E and F ), and SK muscle ( H and I ). Fluorescence of GFP-GABARAP was observed under confocal laser-scanning microscopy (FV1000: Olympus), and GFP-GABARAP dots were counted using an ImageJ program ( http://rsbweb.nih.gov/ij/ ) with a TopHat plugin ( http://rsb.info.nih.gov/ij/plugins/lipschitz/ ). ( D , G , and J ) Relative ratios of GFP-GABARAP dots per unit area in the heart ( D ), liver ( E ), and skeletal muscle ( J ), using at least 10 images from each tissue in four mice. Fed , tissues under fed conditions; Starvation , tissues under starvation conditions for 48 h. Bars indicate 10 mm.

Article Snippet: Polyclonal anti-GFP antibody was purchased from Rockland (600-101-215).

Techniques: Expressing, Transgenic Assay, Control, Fluorescence, Confocal Laser Scanning Microscopy

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) HEK 293T cells expressing FLAG-STX17 were infected with different strains of Legionella for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A), followed by western blots with antibodies against FLAG, GST, and ubiquitin. Whole-cell lysates were blotted with antibodies against FLAG and vinculin as a loading control. This experiment was repeated three times with similar results. ( B ) HEK 293T cells expressing V5-SNAP29 were infected with different Legionella strains for 2 h. Lysates were used for GST pulldown with the DupA trapping mutant GST-DupA (H67A) followed by western blotting with antibodies against V5, GST, and ubiquitin. Whole-cell lysates were probed with antibodies against V5 and vinculin as a loading control. This experiment was repeated three times with similar results. ( C ) Domain architecture of STX17 and SNAP29 showing the PR-ubiquitination sites. ( D ) A549 cells expressing GFP-tagged STX17, STX17TM or the PR-Ub deficient mutant of STX17 were infected with Legionella strains for 2 h before fixation and immunostaining with a Legionella -specific antibody for analysis by confocal microscopy. Control cells were treated with 300 nM Torin-1 for 4 h. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. Scale bar: 5 µm. Scale bar in inset: 2 µm. ( E ) The number of STX17 + bacteria per cell were counted for ~50 cells taken three different experiments. In the box plots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. N = 52, 56 cells taken from three experimental replicates. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 7.43E-8. Scale bar: 5 µm. ( F ) The formation of WT and PR-Ub-deficient SNAP29-GFP puncta was monitored in Legionella -infected cells 4 h post-infection. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) SNAP29 puncta were counted in 50-μm 2 regions of interest using FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 50 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, For the graph SNAP29 puncta/cell: *** P = 6.82E-20. For the graph % cells with SNAP29 recruitment to bacteria: *** P = 2.27E-5 (WT, SNAP29WT vs ΔS, SNAP29WT) *** P = 2.56E-6 (WT, SNAP29WT vs WT, SNAP29mut. Scale bar: 5 µm. .

Article Snippet: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

Techniques: Expressing, Infection, Mutagenesis, Western Blot, Ubiquitin Proteomics, Control, Immunostaining, Confocal Microscopy, Bacteria, Software, Two Tailed Test

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) (i) HEK 293 T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. FLAG-STX17 was immunoprecipitated using FLAG resin and analyzed by western blot using antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated 2 times with similar results. (ii) HEK 293T cells were cotransfected with FLAG-STX17 and GFP-tagged SdeA/SdeA(EE/AA) or a control vector and immunoprecipitated as shown in ( B ). The samples were then treated with or without pure DupA for 1 h before western blotting with antibodies against FLAG to detect PR-Ub-modified and unmodified FLAG-STX17. The experiment was repeated two times with similar results. ( B ) HEK 293T cells were cotransfected with GFP-SNAP29 and HA-tagged SdeA/SdeA(EE/AA) or a control vector for 16 h. GFP-SNAP29 was immunoprecipitated with anti-GFP beads, treated with or without pure DupA for 1 h and analyzed by western blot with antibodies against GFP to detect PR-Ub-modified and unmodified SNAP29. The experiment was repeated two times with similar results. ( C ) GST-STX17 and GST-STX17(S195AS202AS209A) were incubated with or without SdeA in the presence of 1 mM NAD + and ubiquitin for 1 h. The samples were analyzed by western blot using antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. ( D ) GST-STX17 and its PR-Ub-deficient mutant (S195AS202AS209A) were modified with or without SdeA, in the presence of 1 mM NAD+ and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A) ( E ) GST-SNAP29 and its PR-Ub-deficient mutant (S61AS63AS70A) were modified with or without SdeA, in the presence of 1 mM NAD + and ubiquitin for 1 h. Samples were analyzed by western blot with antibodies against ubiquitin and GST to detect PR-Ub. The experiment was repeated three times with similar results. SdeA(EE/AA): mART mutant SdeA(E860AS862A).

Article Snippet: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

Techniques: Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Modification, Incubation, Ubiquitin Proteomics, Mutagenesis

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) Mass spectrum and deduced sequence map of PR-Ub-modified SNAP29. ( B ) HEK 293 T cells were transfected with GFP-tagged WT STX17, STX17TM, or the STX17 serine mutant (S195AS202AS209A) followed by Legionella infection for 2 h. STX17 was then immunoprecipitated using anti-GFP beads followed by western blotting with antibodies against GFP and ubiquitin. Cell lysates were analyzed by western blot with an antibody against GFP to check the expression levels of GFP-STX17 constructs. The experiment was repeated three times with similar results.

Article Snippet: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

Techniques: Sequencing, Modification, Transfection, Mutagenesis, Infection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Construct

Journal: The EMBO Journal

Article Title: Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection

doi: 10.1038/s44318-025-00483-4

Figure Lengend Snippet: ( A ) A549 cells expressing STX17-GFP were infected with WT Legionella for 2 h in the presence or absence of 100 nM brefeldin A before fixation and staining the intracellular bacteria by DAPI. STX17-positive bacteria was counted in 50 cells per set, taken from three independent experiments. Error bars indicate SEM. Difference between sets was non-significant from p value calculated by two-tailed, type 3 Student’s t test. Scale bar:10 µm. ( B ) A549 cells expressing STX17-GFP were infected with Legionella (WT/ΔS) for 2 h in the presence or absence of 100 nM wortmannin before fixation and immunostaining with antibodies against Legionella . p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.45E-6(WT and ΔS sets without wortmannin), *** P = 2.05E-5 (WT +/-wortmannin), Graph represents n = 50 cells taken from three experiments, error bars indicate SEM. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( C ) Immuno-electron microscopy of HeLa cells transfected with STX17-GFP and infected with WT Legionella-DsRed for 4 h. Ultrathin cryosection immunogold labeled for STX17-GFP by protein A–10-nm gold. Colors are added by Photoshop: Yellow marks a STX-17.GFP-positive ER cisterna closely aligned with the Legionella (Leg) containing vacuole. Green marks the space between the vacuolar membrane and enclosed Legionella. Bar, 200 nm. ( D ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the STX17 and Legionella antibodies to check for the recruitment of STX17 to intracellular bacteria. White arrows mark intracellular bacteria with STX17 recruitment. The data are means ± SEM of 118 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 4.21E-6. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( E ) A549 cells were infected with WT or ΔRΔS Legionella for 1 h, fixed and immunostained with the SNAP29 and Legionella antibodies to check for the recruitment of SNAP29 to intracellular bacteria. White arrows mark intracellular bacteria with SNAP29 recruitment. The data are means ± SEM of 120 cells from three independent experiments. p value was calculated by two-tailed, type 3 Student’s t test. *** P = 2.21E-4. Scale bar: 5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( F ) HeLa cells were cotransfected with RFP-tagged SdeA or its catalytic mutant (E860AE862A) and GFP-tagged WT SNAP29 or its PR-Ub-deficient mutant. Cells were treated with 300 nM Torin-1 for 4 h to induce autophagy before fixation and confocal imaging. Scale bar:5 µm. Dotted lines indicate cell outlines drawn from thresholding images in FIJI. ( G ) The graph shows the number of cells with SNAP29-GFP puncta (from panel d) counted in FIJI. In the box plot, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n > 30 cells taken from three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 0.00032. In bar graph, the data are means ± SEM of n > 30 cells from three independent experiments. Scale bar:5 µm. ( H ) A549 cells were treated with SNAP29 or control siRNA for 48 h followed by infection Legionella. Intracellular bacterial replication was assessed after 0, 24 and 48 h. Data are means ± SEM of three independent experiments. P value was calculated using two-tailed, type 3 Student’s t test, *** P = 2.1E-4 (ΔR), ** P = 0,.031(ΔRΔS). (ni not infected, WT wild-type, Legionella , ΔS-ΔSidE Legionella ).

Article Snippet: We used the following antibodies and dilutions: STX17 (cat. no. 17815-1-AP, Proteintech; 1:1000), GAPDH (cat. no. 2118, Cell Signaling Technology; 1:2000), GFP trap beads (cat. no. gta-100, ChromoTek), GFP (cat. no. sc-9996, Santa Cruz Biotechnology; 1:2000), GFP for immune-electron microscopy (cat no. 600–106-215, Rockland), biotin for immuno-electron microscopy (cat. no. 100–4198, Rockland), LC3 (cat. no. 2775, Cell Signaling Technology; 1:2000), VAMP8 (cat. no. 13060, Cell Signaling Technology; 1:1000), SNAP29 (cat. no. 3013, Cell Signaling Technology; 1:2000), RAB5 (cat. no. 3547; 1:1,000), RAB7 (cat. no. 9367, Cell Signaling Technology; 1:2000), ATG16L (cat. no. ab188642, Cell Signaling Technology; 1:1000), ATG12 (cat. no. 4180, Cell Signaling Technology; 1:1000), Beclin1 (cat. no. 3738, Cell Signaling Technology; 1:1000), FIP200 (cat. no. 17250-1AP, Proteintech; 1:1000), LAMP1 (cat. no. 9091, Cell Signaling Technology; 1:2000), and Legionella (cat. no. 20943, Abcam; 1:4000), Ubiquitin (Cat. no: 3933, Cell Signaling Technology, 1:1000).

Techniques: Expressing, Infection, Staining, Bacteria, Two Tailed Test, Immunostaining, Immuno-Electron Microscopy, Transfection, Labeling, Membrane, Mutagenesis, Imaging, Software, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: PLoS Pathogens

Article Title: Rotavirus NSP1 Inhibits NFκB Activation by Inducing Proteasome-Dependent Degradation of β-TrCP: A Novel Mechanism of IFN Antagonism

doi: 10.1371/journal.ppat.1000280

Figure Lengend Snippet: (A) MA104 cells were infected with three pfu/cell of the indicated virus strain. Lysates were prepared at 2, 4, 6, 8, and 10 hpi and the abundance of p65 was determined by immunoblot using anti-p65 antibody. Blots were probed with anti-GAPDH as a loading control. (B) The subcellular localization of p65 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined at six hpi by confocal microscopy (63×, NA 1.40). Cells were stained with anti-p65 antibody, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG.

Article Snippet: The primary antibodies were mouse anti-IRF3 (Santa Cruz Biotechnology) or rabbit anti-p65 (Rockland).

Techniques: Infection, Virus, Western Blot, Control, Confocal Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Rotavirus NSP1 Inhibits NFκB Activation by Inducing Proteasome-Dependent Degradation of β-TrCP: A Novel Mechanism of IFN Antagonism

doi: 10.1371/journal.ppat.1000280

Figure Lengend Snippet: MA104 cells were infected with A5-16, OSU or NCDV at an moi of three pfu/cell. Six hours post-infection, nuclear and cytoplasmic fractions were separated with the nuclear extract kit following the manufacturer's instructions (Active Motif). (A) p65 activation measured by p65 TransAm ELISA. Error bars are the standard error of the mean. (B) Nuclear fractions were probed with anti-p65 antibody, anti-laminA/C (nuclear, BD Biosciences), and anti- GAPDH (cytoplasmic) antibodies.

Article Snippet: The primary antibodies were mouse anti-IRF3 (Santa Cruz Biotechnology) or rabbit anti-p65 (Rockland).

Techniques: Infection, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of cell science

Article Title: Nuclear import of hTERT requires a bipartite nuclear localization signal and Akt-mediated phosphorylation.

doi: 10.1242/jcs.099267

Figure Lengend Snippet: Fig. 5. Inhibition of Akt activity attenuates nuclear translocation of endogenous hTERT. (A) H1299 and HT1080 cells were transfected with HA-tagged dominant negative Akt (DN-Akt) and analyzed by immunofluorescence staining with anti-hTERT (red) or anti-HA (green) antibodies. The nuclei were stained with DAPI (blue). (B) After transfection, cells were analyzed quantitatively for the cellular location of endogenous hTERT. (C) H1299 and HT1080 cells were treated with 1 mM wortmannin, or left untreated, for 1 hour and then subjected to immunofluorescence staining with anti-hTERT antibody (green). (D) H1299 and HT1080 cells were treated with 1 mM wortmannin for 1 hour and analyzed quantitatively for the cellular location of endogenous hTERT.

Article Snippet: To detect endogenous hTERT, cells were incubated with rabbit anti-hTERT antibody (Rockland Immunochemicals, Gilbertsville, PA) overnight at 4 C̊, followed by incubation with either FITCconjugated anti-rabbit immunoglobulin or Rhodamine-conjugated anti-rabbit immunoglobulin (Invitrogen, Carlsbad, CA).

Techniques: Inhibition, Activity Assay, Translocation Assay, Transfection, Dominant Negative Mutation, Immunofluorescence, Staining

Journal: Journal of cell science

Article Title: Nuclear import of hTERT requires a bipartite nuclear localization signal and Akt-mediated phosphorylation.

doi: 10.1242/jcs.099267

Figure Lengend Snippet: Fig. 8. hTERT expression alleviates basal intracellular ROS levels. (A) HFF cells stably expressing hTERT or S227A/7A at ,40 PDs after selection were analyzed by indirect immunofluorescence with anti-FLAG (green) and anti-Tom20 (red) antibodies. DNA was stained with DAPI (blue). (B,C) For detection of the intracellular ROS levels, HFF cells stably expressing the empty vector, hTERT or S227A/7A at ,70 PDs were incubated with either 10 mM CM-DCFH-DA (B) or 5 mM MitoSOX RED (C) and analyzed by flow cytometry, as described in the Materials and Methods. The average G-values were computed, and statistical analyses were performed using a two-tailed Student’s t-test (*P,0.005). (D,E) For fluorescence detection of the intracellular ROS levels, HFF cells stably expressing the empty vector, hTERT or S227A/7A at ,70 PDs were incubated with 10 mM DCFH-DA and 5 mM MitoSOX RED. Digital images of DCF (D) and MitoSOX (E) fluorescence were obtained by confocal microscopy and overlaid on the DAPI images. The mean fluorescence intensity per image was calculated using MetaMorph software and averaged for ten images. Statistical analyses were performed using a two-tailed Student’s t-test (*P,0.01).

Article Snippet: To detect endogenous hTERT, cells were incubated with rabbit anti-hTERT antibody (Rockland Immunochemicals, Gilbertsville, PA) overnight at 4 C̊, followed by incubation with either FITCconjugated anti-rabbit immunoglobulin or Rhodamine-conjugated anti-rabbit immunoglobulin (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Stable Transfection, Selection, Immunofluorescence, Staining, Plasmid Preparation, Incubation, Flow Cytometry, Two Tailed Test, Fluorescence, Confocal Microscopy, Software

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Nemaline bodies form in skeletal muscle in Tg(ACTA1 D286G - eGFP) high zebrafish. Nemaline bodies were detected in a skeletal muscle ( arrowheads ) and pectoral fins ( arrow ) ( i and ii overlaid with brightfield), b heart ( arrowheads ) ( i and ii overlaid with brightfield), c facial muscles ( arrowheads ) ( i and ii overlaid with brightfield) and d ocular muscles ( arrowheads ; i and overlaid with ii brightfield)

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Muscles

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Characterization of skeletal muscle pathology in Tg(ACTA1 D286G - eGFP) high zebrafish. a Skeletal muscle expressing i mosaic ACTA1 D286G -eGFP, and ii overlaid with a light microscopy image of the same section showing Gomori trichrome staining, and iii enlarged. Dark regions (indicative of nemaline bodies) of disrupted muscle correspond to eGFP expression ( arrows ). b Correlative light and electron microscopy of Tg(ACTA1 D286G - eGFP) high fish muscle at 2 dpf. b i Fluorescent image and corresponding ii electron microscopy image of skeletal muscle section containing a dense, elongated nemaline body ( arrow ) and enlarged in ( iii ). Sections are matched using nuclei positions ( asterisk , plus and hash ). c i Accumulations of actin filaments ( arrowheads ) and ii diffuse regions of filamentous actin ( arrowheads ), as well as ii disrupted sarcomeric regions are evident in Tg(ACTA1 D286G - eGFP) high skeletal muscle, at 2 dpf unlike the iii uniform sarcomeres observed in Tg(ACTA1 - eGFP) zebrafish

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Expressing, Light Microscopy, Staining, Electron Microscopy

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Formation of nemaline bodies and aggregates in Tg(ACTA1 D286G - eGFP) high zebrafish. a Maximum projection images from time lapse of Tg(ACTA1 D286G - eGFP) high fish from 56 to 71 hpf showing nemaline bodies distributed throughout the skeletal muscle ( arrows ). Nemaline bodies’ fragment from 59.5 hpf ( arrows ), coincident with the formation of aggregates at the myosepta ( arrowheads ). b Quantification of the mean number of nemaline bodies in Tg(ACTA1 D286G - eGFP) low ( n = 50 per stage) and Tg(ACTA1 D286G - eGFP) ( n = 48 per stage) strains. c Quantification of the percentage of fish displaying globular aggregates in Tg(ACTA1 D286G - eGFP) low and Tg(ACTA1 D286G - eGFP) high strains ( n = 50 per stage). Error bars represent SEM from three independent experiments ( n = 45 per replicate)

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques:

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Quantification of muscle function in Tg(ACTA1 - eGFP) zebrafish. a Quantification of the maximum acceleration recorded from touch-evoked response assays of Tg(ACTA1 D286G - eGFP) high and Tg(ACTA1 wildtype - eGFP) high zebrafish compared to control zebrafish at 2 dpf. Error bars represent SD for n = 15–19 zebrafish, * p < 0.05. b qRT-PCR analysis of ACTA1 - eGFP expression in transgenic lines at 2 dpf. No significant difference was observed between Tg(ACTA1 D286G - eGFP) high and Tg(ACTA1 wildtype - eGFP) high zebrafish. Error bars represent ±SEM for four replicate experiments with each experiment comprising a pooled samples of 20 fish, * p < 0.05, ** p < 0.01. c , d Quantification of the c number of small movements and d distance traveled by Tg(ACTA1 D286G - eGFP) high and Tg(ACTA1 wildtype - eGFP) high and Tg(ACTA1 D286G - eGFP) low and Tg(ACTA1 wildtype - eGFP) low strains compared to control fish at 6 dpf. Error bars represent ±SEM for three replicate experiments ( n = 48 per experiment), * p < 0.05, ** p < 0.01

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Control, Quantitative RT-PCR, Expressing, Transgenic Assay

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Characterization of nemaline bodies and aggregates in ACTA1-eGFP D286G muscle in zebrafish. At 2 dpf, mosaic expression of ACTA1 D286G -eGFP in the muscle ( green ) results in the formation of nemaline bodies ( arrows ; i , iv , vii ) that do not stain with an actinin2 antibody ( red ; ii and overlaid in iii ), actinin3 antibody ( red ; v and overlaid in vi ), or phalloidin (labeling F-actin, red ; viii and overlaid in ix ) despite correct localization of these markers in the sarcomere. At 4 dpf, mosaic expression of ACTA1 D286G -eGFP results in the formation of globular aggregates ( arrowheads ; i , iv , vii ) in the muscle ( green ) stain with an actinin2 antibody ( red ; ii and overlaid in iii ), actinin3 antibody ( red ; v and overlaid in vi ) and phalloidin ( red ; viii and overlaid in ix )

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Expressing, Staining, Labeling

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: a Maximum projection confocal microscopy images of Tg( ACTA1 wildtype - eGFP ) low and Tg( ACTA1 wildtype - eGFP ) high zebrafish strains injected with two different Nebulin (Neb) morpholinos compared to control uninjected embryos at 2 dpf. There is an increased prevalence of eGFP-positive globular aggregates at the myosepta ( arrowheads ) in Tg( ACTA1 wildtype - eGFP ) high Neb morphants compared to controls. Knockdown of Neb produces globular aggregates at the myosepta ( arrowheads ) in Tg( ACTA1 wildtype - eGFP ) low Neb morphants that are absent in control uninjected embryos. b Maximum projection confocal microscopy images of wild-type embryos injected with Neb morpholinos at 2 dpf and stained with phalloidin shows an increase in actin-positive aggregates at the myosepta ( arrowheads ), which are absent in control uninjected embryos

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Confocal Microscopy, Injection, Control, Knockdown, Staining

Journal: Acta Neuropathologica

Article Title: Zebrafish models for nemaline myopathy reveal a spectrum of nemaline bodies contributing to reduced muscle function

doi: 10.1007/s00401-015-1430-3

Figure Lengend Snippet: Fluorescence recovery after photobleaching (FRAP) analyses of ACTA1 and ACTA1 D286G . a Confocal images of ACTA1-eGFP localization at the Z-disk ( white boxes ) and along the thin filament ( yellow boxes ) in single muscle fibers of Tg(ACTA1 D286G - eGFP) high and Tg(ACTA1 wildtype - eGFP) high embryos at 2 dpf. Image sequence shows ACTA1-eGFP prior to photobleaching (pre-bleach), at the time of photobleaching (bleach, 0 s), and 60, 300 and 600 s following photobleaching. Prior to photobleaching eGFP in Tg(ACTA1 wildtype - eGFP) high muscle is primarily localized to the Z-disk ( white boxes ), whereas in Tg(ACTA1 D286G - eGFP) high fibers, eGFP expression is more diffuse throughout the filament ( yellow boxes ). b Quantification of the fluorescence intensity at the Z-disk compared to the filament in Tg(ACTA1 D286G - eGFP) high and Tg(ACTA1 wildtype - eGFP) high muscle fibers. Error bars represent SD for 12 animals (quantifying 2 fibers per animal), *** p < 0.001. c Recovery profiles for ACTA1-eGFP and ACTA1 D286G -eGFP at the Z-disk and filament. Error bars represent SD for 8–10 animals (quantifying 2 fibers per animal)

Article Snippet: The combined immunofluorescence labeling was carried out as described [ ] using a biotinylated anti-eGFP antibody (1:300, Rockland #600-106-215), rabbit anti-biotin (1:10.000, Rockland #100-4198), and a goat anti-rabbit AlexaFluorTM-488-labeled secondary antibody (Molecular Probes, 1:300).

Techniques: Fluorescence, Sequencing, Expressing

Journal: Scientific Reports

Article Title: A major allogenic leukocyte antigen in the agnathan hagfish

doi: 10.1038/srep01716

Figure Lengend Snippet: (A) Flow cytometric fractionation of peripheral blood cells.Forward vs. 90° light scattering profiles are shown. Giemsa staining of the fractionated cells is shown on the right. Fractions R1, R2, and R3 contained myeloid cells, lymphocytes and erythrocytes, respectively. Scale bars indicate 10 μm. (B) Flow cytometric analysis of NICIR3/ALA expression in fractions, R1–R3. NICIR3/ALA is detected in R1 (myeloid cells) and R2 (lymphocytes), but not in R3 (erythrocytes). Cells stained with pre-immune serum was used as a negative control; n = 8; *, P < 0.05 and **, P < 0.01 from Welch's t-test. (C) Immunostaining of buffy coat leukocytes with antibodies against VLR-A or VLR-B (green) and NICIR3/ALA (red). NICIR3/ALA was expressed in VLR-B positive cells more than in VLR-A cells. n = 4; ***, P < 0.001 from Welch's t-test. Scale bars indicate 50 μm. (D) Confocal microscopy analysis of NICIR3/ALA -positive cells. One day after the injection of KLH, buffy coat leukocytes were stained with anti-NICIR3/ALA and anti-KLH antibodies. KLH (green) colocalized with the NICIR3/ALA staining (red) in leukocytes. n = 3. A scale bar is 2 μm. (E) Colocalization of KLH and NICIR3/ALA in a phagocytic leukocyte by immunoelectron microscopy. As in (D), buffy coat leukocytes were stained with anti-NICIR3/ALA and anti-KLH antibodies; n = 3. Two representative data are shown. NICIR3/ALA and KLH were labeled with 30 nm and 6 nm gold particles, respectively. Enlarged views are shown on the right. Scale bars indicate 100 nm.

Article Snippet: The cells were fixed with 4% PFA for 1 hr and incubated at room temperature for 2 hrs with rabbit anti-KLH antibodies (Rockland) or anti-OVA antibodies (Delta Biolabs), and with mouse anti- NICIR3/ALA antibodies.

Techniques: Fractionation, Staining, Expressing, Negative Control, Immunostaining, Confocal Microscopy, Injection, Immuno-Electron Microscopy, Labeling