Journal: eLife

Article Title: Endocytic recycling and vesicular transport systems mediate transcytosis of Leptospira interrogans across cell monolayer

doi: 10.7554/eLife.44594

Figure Lengend Snippet:

Article Snippet: Using rat anti-strain Lai-IgG, rabbit anti-Rab5 or Rab11-IgG (Cell Signaling) or goat anti-transferrin receptor (TfR)-IgG (Santa Crus) as the primary antibody, AlexaFluor594-conjugated donkey anti-rat-IgG, AlexaFluor488-conjugated donkey anti-rabbit or goat-IgG or AlexaFluor405-conjugated donkey anti-rabbit-IgG (Abcam) as the second antibody, Lep-vesicle-Rab5 or Lep-vesicle-Rab11/TfR co-localization were detected by confocal microscopy (402/421 nm excitation/emission wavelengths for AlexaFluor405 detection) and the yellow or white FI reflecting the co-localization was measured as above.

Techniques: Control, Fluorescence, Western Blot, Bicinchoninic Acid Protein Assay, Negative Control

Journal: Journal of neuroscience methods

Article Title: A white matter stroke model in the mouse: Axonal damage, progenitor responses and MRI correlates

doi: 10.1016/j.jneumeth.2009.03.017

Figure Lengend Snippet: Cell death in white matter one day after stroke. (A) Immunohistochemical staining for AIF in contralateral hemisphere. (B) AIF stain in the hemisphere ipsilateral to stroke. Note positive cells in white matter and deepest cortical layers, and linear rows of AIF immunoreactive cells (arrows). (C) CAII stain for oligodendrocytes (green) activated PARP immunoreactivity (red). Note multiple CAII and activated PARP double positive cells in subcortical white matter (D) In situ nick translation showing double stranded DNA nicking in apoptotic cells in subcortical white matter. (E) Transferrin immunoreactive oligodendrocytes in control subcortical white matter region. (F) Transferrin immunoreactive oligodendrocytes 7 days after subcortical white matter stroke. (G) NG2 immunoreactive OPCs in control subcortical white matter region. (H) NG2 immunoreactive OPCs 14 days after subcortical white matter stroke. ctx = cortex, str = striatum, wm = subcortical white matter. Bar in (B) = 50 μm and applies to (A and B); bar in (Cand D) = 40 μm, Bar in (F) = 20 μm and applies to (E–H) (for interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).

Article Snippet: Primary antibodies were: rat anti-myelin basic protein (1:800, Carlsbad, CA), rabbit anti-myelin basic protein (MBP)(1:200, Abcam, Cambridge, MA), mouse anti-NeuN (1:500, Abcam), rat anti-GFAP (1:1000, Invitrogen), sheep anti-carbonic anhydrase II (1:600, Morphosys, Germany), sheep and rat anti-BrdU (1:500, Abcam), rabbit anti-transferrin (1:75, Abcam), mouse anti-apoptosis inducing factor (1:200, AIF, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-activated Poly(ADP-ribose)polymerase 1 (PARP, 1:100, Cell Signaling, Danvers, MA), rabbit anti-NF200 (1:2000, Sigma, St. Louis, MO), mouse anti-SMI31 and anti-SMI-32 (1:1000, Abcam), rabbit anti-NG2 (1:200, Invitrogen); goat anti-transferrin (1:100, MP Biomedicals, Solon, OH), rabbit anti-IBA1 (1:1000, Wako Chemicals, Richmond, VA) and NeuN (1:500).

Techniques: Immunohistochemical staining, Staining, In Situ, Nick Translation

Journal: Journal of neuroscience methods

Article Title: A white matter stroke model in the mouse: Axonal damage, progenitor responses and MRI correlates

doi: 10.1016/j.jneumeth.2009.03.017

Figure Lengend Snippet: Early and late response of OPCs and oligodendrocytes following subcortical stroke. BrdU pulse-chase experiment at day 7 and 28 after subcortical stroke. (A) Representative images of BrdU double labeling of oligodendrocyte progenitor cells (OPC) with OPC marker NG2 (top panel), and oligodendrocytes with specific marker transferrin (bottom panel) at day 7. The insets correspond to higher power views. Bar in right middle panel of A = 20 μm. (B) Total number of double labeled cells estimated by stereology: means ± standard deviations. NG2 7 day vs. 28 day 3918.33 ± 238.03, 2150.0 ± 413.54, p = 0.003; transferrin 7 day vs. 28 day 3665.0 ± 456.54, 1941.67 ± 677.98, p = 0.02.

Article Snippet: Primary antibodies were: rat anti-myelin basic protein (1:800, Carlsbad, CA), rabbit anti-myelin basic protein (MBP)(1:200, Abcam, Cambridge, MA), mouse anti-NeuN (1:500, Abcam), rat anti-GFAP (1:1000, Invitrogen), sheep anti-carbonic anhydrase II (1:600, Morphosys, Germany), sheep and rat anti-BrdU (1:500, Abcam), rabbit anti-transferrin (1:75, Abcam), mouse anti-apoptosis inducing factor (1:200, AIF, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-activated Poly(ADP-ribose)polymerase 1 (PARP, 1:100, Cell Signaling, Danvers, MA), rabbit anti-NF200 (1:2000, Sigma, St. Louis, MO), mouse anti-SMI31 and anti-SMI-32 (1:1000, Abcam), rabbit anti-NG2 (1:200, Invitrogen); goat anti-transferrin (1:100, MP Biomedicals, Solon, OH), rabbit anti-IBA1 (1:1000, Wako Chemicals, Richmond, VA) and NeuN (1:500).

Techniques: Pulse Chase, Labeling, Marker

Journal: PLoS ONE

Article Title: Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

doi: 10.1371/journal.pone.0064975

Figure Lengend Snippet: A , Schematic representation of the anti-pS324/325, anti-pS338/339, anti-pS346/347, and anti-S341-S352 (UMB-2) antibodies with their epitopes in the human CXCR4 receptor. B–G , HEK293 cells stably transfected with hemagglutinin (HA) epitope-tagged CXCR4 received CXCL12 (20 nM) or vehicle for 15 min and were lysed. An aliquot of the lysate from stimulated cells was dephosphorylated using Lambda-Protein Phosphatase (λ-PP). Samples were separated on 10% SDS polyacrylamide gels, blotted, and detected with anti-S341-S352 (B), anti-pS346/347 (C), anti-pS338/339 (D), anti-pS324/325 (E), anti-HA-tag (F), and anti-transferrin receptor (TFR) (G). B , CXCL12 stimulation causes a strong loss of anti-S341-S352 binding; dephosphorylation restores antibody binding. C–E , The phospho-selective antibodies recognize CXCR4 only in the non-dephosphorylated sample from stimulated cells. Anti-pS324/325 produces a non-specific band at approximately 70 kDa (arrowhead in E). H , HEK293 cells transiently transfected with a HA-CXCR4-ST/A mutant (all serines and threonines in the C-terminal domain were converted into alanines) were stimulated with CXCL12. Four aliquots were loaded in a Western blot and separately detected with the indicated antibodies. The phospho-selective antibodies do not detect the CXCL12-stimulated phosphorylation-deficient CXCR4 mutant (the arrow points to 47 kDa which corresponds to the size of CXCR4). F , G , H , Equal loading was controlled in stripped blots by detecting the HA-tag of recombinant expressed CXCR4 and by detecting endogenous TFR.

Article Snippet: Blots were immunodetected using anti-CXCR4 (UMB-2), anti-pS338/339 (#3152), anti-pS346/347 (#3208), anti-pS324/325 (#5199), anti-HA-tag (#631), anti-T7-tag or anti-transferrin receptor antibodies and appropriate peroxidase-conjugated secondary antibodies (#sc2004, Santa Cruz Biotechnology Inc., Heidelberg, Germany; #NXA931 GE Healthcare).

Techniques: Stable Transfection, Transfection, Binding Assay, De-Phosphorylation Assay, Mutagenesis, Western Blot, Phospho-proteomics, Recombinant

Journal: PLoS ONE

Article Title: Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

doi: 10.1371/journal.pone.0064975

Figure Lengend Snippet: Sequential phosphorylation at CXCR4 C-terminal sites. A–F , Stably CXCR4-transfected HEK293 cells were stimulated with 20 nM CXCL12 for 0, 2, 5, and 15 min. Aliquots of the lysates were detected in four immunoblots using the indicated antibodies. Blots were stripped and reprobed with anti-HA and anti-transferrin receptor (TFR) to control for equal loading. A , B , CXCL12 induces a rapid loss of the anti-S341-S352 signal and a concomitant immediate increase of the anti-pS346/347 signal. C , Maximal S338/339 phosphorylation requires 15 min CXCL12 stimulation. D , Demonstration of rapid CXCL12-promoted S324/325 phosphorylation. E – I , The signal ratio versus anti-HA was determined for the four anti-CXCR4 antibodies using densitometry. The ratio was set as 100% at 0 min for anti-S341-S352 (G) and at 15 min for the phospho-selective antibodies (G–I). Data represent mean+SEM from 4–5 independent experiments. G , Note complementarity of the anti-S341-S352 and anti-pS346/347 time courses. H , I , Phosphorylation occurs faster at S346/347 than at S338/339 and S324/325. *: p<0.05; 1way ANOVA and Bonferronís multiple comparison post-test for selected groups.

Article Snippet: Blots were immunodetected using anti-CXCR4 (UMB-2), anti-pS338/339 (#3152), anti-pS346/347 (#3208), anti-pS324/325 (#5199), anti-HA-tag (#631), anti-T7-tag or anti-transferrin receptor antibodies and appropriate peroxidase-conjugated secondary antibodies (#sc2004, Santa Cruz Biotechnology Inc., Heidelberg, Germany; #NXA931 GE Healthcare).

Techniques: Phospho-proteomics, Stable Transfection, Transfection, Western Blot, Control, Comparison

Journal: PLoS ONE

Article Title: Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

doi: 10.1371/journal.pone.0064975

Figure Lengend Snippet: A–E , Stably CXCR4-transfected HEK293 cells were stimulated with CXCL12 for 30 min, washed with acidic buffer, and incubated with AMD3100-supplemented medium for the indicated intervals. Aliquots of the lysates were detected in five immunoblots using the indicated antibodies. The HA-tag and the endogenous transferrin receptor (TFR) were detected as loading controls. A , B , Slow recovery of the anti-S341-S352 signal corresponds to long-lasting phosphorylation at S346/347. C , D , Phosphorylation at S338/339 and S324/325 is rapidly reversed after CXCL12 washout. E , Detection with anti-HA demonstrates similar CXCR4 levels in the different samples. F–I , Using densitometry, the signal ratio versus anti-HA was determined for the four phospho-sensitive antibodies. Non-stimulated samples were set as 100% in F and samples undergoing 30 min CXCL12 stimulation were set as 100% in G–I . While there is little dephosphorylation at S346/347 (F,G ) , major dephosphorylation occurs already 15 min after CXCL12 washout at S338/339 and S324/324 (H,I). Data represent mean+SEM from 4–5 independent experiments. *: p<0.05; 1way ANOVA followed by Dunnett´s post-test vs. the 30 min CXCL12/0 min washout group.

Article Snippet: Blots were immunodetected using anti-CXCR4 (UMB-2), anti-pS338/339 (#3152), anti-pS346/347 (#3208), anti-pS324/325 (#5199), anti-HA-tag (#631), anti-T7-tag or anti-transferrin receptor antibodies and appropriate peroxidase-conjugated secondary antibodies (#sc2004, Santa Cruz Biotechnology Inc., Heidelberg, Germany; #NXA931 GE Healthcare).

Techniques: Stable Transfection, Transfection, Incubation, Western Blot, Phospho-proteomics, De-Phosphorylation Assay

Journal: PLoS ONE

Article Title: Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

doi: 10.1371/journal.pone.0064975

Figure Lengend Snippet: HEK293 cells were transiently transfected with N-terminal T7 epitope-tagged wildtype CXCR4 (T7-CXCR4) and C-terminal deletion mutants lacking the last 10 and 17 residues (T7-CXCR4-Δ10, T7-CXCR4-Δ17). A–F , Surface receptors were pulse-labeled with anti-T7 antibody in live cells before cells received vehicle (A–C) or CXCL12 (D–F) for 30 min. Cells were fixed and permeabilized before immunofluorescent detection of anti-T7. In the absence of CXCL12 all three receptors are targeted to the plasma membrane showing little internalization (A–C). CXCL12 causes strong internalization of T7-CXCR4 (D) but only weak internalization of the C-terminal truncated receptors (E,F). G , Western blot analyses of CXCL12-induced phosphorylation of T7-CXCR4, T7-CXCR4-Δ10, and T7-CXCR4-Δ17 (CXCR4, Δ10, Δ17). HEK293 cells were transiently transfected with the indicated constructs and harvested before or 15 min after CXCL12 stimulation. Aliquots of the lysates were dephosphorylated using Lambda-Protein Phosphatase (λ-PP). Samples were loaded in four SDS gels, blotted, and immunodetected using anti-S341-S352, anti-pS346/347, anti-pS338/339, and anti-pS324/325. T7-CXCR4: In samples from non-stimulated transfectants λ-PP-treatment slightly enhances the signal of anti-S341-S352 and almost eliminates signals of the phospho-selective antibodies, indicating some constitutive CXCR4 phosphorylation. CXCL12 treatment causes a strong signal reduction of anti-S341-S352 and a strong signal increase of anti-pS346/347, anti-pS338/339, and anti-pS324/325. λ-PP-treatment of stimulated lysates fully restores the anti-S341-S352 signal and eliminates the signal of the phospho-selective antibodies. T7-CXCR4- Δ 10: Deletion of the epitope eliminates specific signals of anti-S341-S352 and anti-pS346/347. There is virtually no constitutive phosphorylation at S338/339 and S324/325 in non-stimulated lysates. CXCL12-induced phosphorylation is not detected at S338/339 and strongly reduced at pS324/325 as compared with T7-CXCR4. T7-CXCR4- Δ 17: Deletion of CXCR4 residues 336–352 eliminates specific signals of anti-S341-S352, anti-pS346/347, and anti-pS338/339. The mutant shows virtually no constitutive and no CXCL12-induced phosphorylation at S324/325. Anti-T7-tag: Stripping and detection with the anti-tag antibody reveals similar CXCR4 expression levels in all samples. Anti-TFR: Detection of endogenous transferrin receptor (TFR) in a stripped blot demonstrates equal loading. G , Results are representative for three independent experiments with similar results.

Article Snippet: Blots were immunodetected using anti-CXCR4 (UMB-2), anti-pS338/339 (#3152), anti-pS346/347 (#3208), anti-pS324/325 (#5199), anti-HA-tag (#631), anti-T7-tag or anti-transferrin receptor antibodies and appropriate peroxidase-conjugated secondary antibodies (#sc2004, Santa Cruz Biotechnology Inc., Heidelberg, Germany; #NXA931 GE Healthcare).

Techniques: Transfection, Labeling, Clinical Proteomics, Membrane, Western Blot, Phospho-proteomics, Construct, Mutagenesis, Stripping Membranes, Expressing

Journal: PLoS ONE

Article Title: Hierarchical Organization of Multi-Site Phosphorylation at the CXCR4 C Terminus

doi: 10.1371/journal.pone.0064975

Figure Lengend Snippet: A , Schematic representation of the HA-CXCR4-S346-8A and HA-CXCR4-E343K mutants. B , C , HEK293 cells were transfected with empty vector (mock) and the indicated CXCR4 mutants. B , The anti-S341-S352 antibody recognizes CXCR4-E343K only slightly and does not bind to CXCR4-S346-8A. C , Cells were stimulated with 20 nM CXCL12 for 30 min. Aliquots of the lysates were detected in four immunoblots using the indicated antibodies. Blots were stripped and reprobed with anti-HA and anti-transferrin receptor (TFR) to control for equal CXCR4 protein and loading. C , CXCR4 mutants are not phosphorylated at S346/347 after CXCL12-stimulation. Constitutive and induced phosphorylation at S338/339 and S324/325 is strongly diminished as compared with wildtype CXCR4. B , C , Results are representative for three independent experiments with similar results.

Article Snippet: Blots were immunodetected using anti-CXCR4 (UMB-2), anti-pS338/339 (#3152), anti-pS346/347 (#3208), anti-pS324/325 (#5199), anti-HA-tag (#631), anti-T7-tag or anti-transferrin receptor antibodies and appropriate peroxidase-conjugated secondary antibodies (#sc2004, Santa Cruz Biotechnology Inc., Heidelberg, Germany; #NXA931 GE Healthcare).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Phospho-proteomics

Journal: Disease Markers

Article Title: Disordered Maternal and Fetal Iron Metabolism Occurs in Preterm Births in Human

doi: 10.1155/2022/1664474

Figure Lengend Snippet: Changes of maternal and cord serum hepcidin and iron regulators in preterm pregnancies. Maternal serum hepcidin at different stages (a), maternal and cord serum hepcidin (b), the PIDI index (c), Western blot analysis of FT-L, FPN, TfR1, and β -actin (d); the quantified data for Western blot results are presented in the right panel, relative REG-1 expression (e). ∗ P < 0.05 and # P < 0.001, compared to the full-term pregnant control or as indicated.

Article Snippet: The membranes were incubated overnight with primary antibodies (Abs) as follows: rabbit anti-ferroportin (FPN) (a generous gift from Prof. Greg Anderson, QIMR Berghofer Medical Research Institute, Australia), rabbit anti- transferrin receptor 1 (TfR1) (Proteintech, China), rabbit anti-ferritin light chain (FT-L) (Proteintech, China), and rabbit anti- β -actin (ABclonal, China).

Techniques: Western Blot, Expressing, Control