Journal: Scientific Reports

Article Title: ATM mediates spermidine-induced mitophagy via PINK1 and Parkin regulation in human fibroblasts

doi: 10.1038/srep24700

Figure Lengend Snippet: The lysates of treated GM00637 cells were immunoblotted with anti-PINK1 or anti-Hsp60 (a loading control of mitochondrial proteins) ( a ). Alternatively, the expression of PINK1 in GM00637 cells was detected by immunofluorescence analysis and visualized with an Axio Observer Z1 fluorescence microscope ( b ). The translocation of Parkin was determined by the expression of Parkin after cell fractionation. GAPDH, a loading control of cytoplasmic proteins; Hsp60 and COX IV, loading controls of mitochondrial proteins. C: cytoplasmic fraction; M: mitochondrial fraction (c). The colocalizations of Parkin with either mitochondria (labeled by COX IV or Hsp60) ( d ), the autophagosomal marker LC3B ( e ), or PINK1 ( f ) were imaged using a Fluoview FV1000 confocal microscope. The length of the scale bar is 10 μm. Bar graphs show the ratios of Parkin/mitochondria, Parkin/LC3B and Parkin/PINK1 colocalizations from 20–45 cells/condition compiled from three experiments. Values are mean ± SD, *p < 0.05 vs. control.

Article Snippet: Briefly, proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, and then incubated with primary rabbit antibodies specific to PINK1 (1:1000, ab23707, polyclonal, Abcam, Cambridge, MA, UK), Parkin (1:1000, A0968, polyclonal, ABclonal, College Park, MD, USA), Hsp60 (1:5000, GTX110089, polyclonal, Genetex Inc., Irvine, CA, USA) and GAPDH (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China) or mouse antibodies specific to COX IV (1:2000, GTX101499, polyclonal, Genetex Inc., Irvine, CA, USA) and β-actin (1:2000, Zhongshan Golden Bridge Biotechnology, Beijing, China).

Techniques: Control, Expressing, Immunofluorescence, Fluorescence, Microscopy, Translocation Assay, Cell Fractionation, Labeling, Marker

Journal: The Journal of Experimental Medicine

Article Title: Cellular Prion Protein Promotes Brucella Infection into Macrophages

doi: 10.1084/jem.20021980

Figure Lengend Snippet: VirB complex–dependent surface expression of immunodominant Hsp60. Immunoblot analysis of whole cell lysates with serum from human brucellosis (A) and of purified Hsp60 with indicated serum (B). (C) Labeling of bacteria grown in vitro with antibody specific for Hsp60. Fluorescence microscopy of stained wild-type, virB2 , or virB4 mutant, and complemented strain for each mutant, with anti-Hsp60 (top) or anti– B. abortus (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (D) Localization of G6PDH on permeabilized B. abortus by immunofluorescence microscopy. Bacteria were probed with anti-G6PDH (top) and stained for DNA with DAPI (middle) in either the absence (−lysozyme) or the presence (+lysozyme) of lysozyme, and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown.

Article Snippet: Anti-Hsp60 rabbit polyclonal antibody was obtained from MBL International Corporation.

Techniques: Expressing, Western Blot, Purification, Labeling, Bacteria, In Vitro, Fluorescence, Microscopy, Staining, Mutagenesis, Immunofluorescence

Journal: The Journal of Experimental Medicine

Article Title: Cellular Prion Protein Promotes Brucella Infection into Macrophages

doi: 10.1084/jem.20021980

Figure Lengend Snippet: Binding of Hsp60 to PrP C . (A) Demonstration of affinity of Hsp60 for PrP C by pull-down assay with Hsp60-coated beads. Control was assessed with beads only, the addition of anti-Hsp60 antibody, or purified Hsp60. Precipitates were analyzed by immunoblotting with anti-PrP C antibody. (B) Cell lysate or immunoprecipitates with anti-PrP C antibody and affinity of PrP C for Hsp60 by pull-down assay with PrP C -coated beads was analyzed by immunoblotting with anti-Hsp60 antibody. Control was assessed with beads only, or the addition of purified PrP C . (C) Silver staining of precipitates by pull-down assay. Samples were purified with Hsp60 (control) and precipitates of pull-down with Hsp60-coated beads (PD). (D) Labeling of internalized bacteria in macrophages with antibody specific for Hsp60. Macrophages were incubated with B. abortus for 5 or 15 min, and Hsp60 were localized by immunofluorescence as described in Materials and Methods. Fluorescence microscopy of stained GFP-expressed wild-type strain with anti-Hsp60 and phase contrast microscopy of the corresponding microscopic fields are shown. Arrows point to bacteria.

Article Snippet: Anti-Hsp60 rabbit polyclonal antibody was obtained from MBL International Corporation.

Techniques: Binding Assay, Pull Down Assay, Control, Purification, Western Blot, Silver Staining, Labeling, Bacteria, Incubation, Immunofluorescence, Fluorescence, Microscopy, Staining

Journal: The Journal of Experimental Medicine

Article Title: Cellular Prion Protein Promotes Brucella Infection into Macrophages

doi: 10.1084/jem.20021980

Figure Lengend Snippet: Aggregation of PrP C by Hsp60 expressed on the surface of L. lactis . Macrophages were incubated with surface Hsp60 + (top) or Hsp60 − (bottom) L. lactis for 5 min, and PrP C was localized by immunofluorescence as described in Materials and Methods. Phase contrast microscopy of the corresponding microscopic fields are shown. Bacteria (shown by arrows) were stained with DAPI. (B) Labeling of L. lactis grown in vitro, with antibody specific for Hsp60. Fluorescence microscopy of stained surface Hsp60 + or Hsp60 − L. lactis with anti-Hsp60 (top) or DAPI (middle) and phase contrast microscopy of the corresponding microscopic fields (bottom) are shown. (C) PrP C -binding activity. Measurement of PrP C -binding activity was performed by ELISA (refer to Materials and Methods). (D and E) Macrophages were incubated with surface Hsp60 + (solid bars) or Hsp60 − (open bars) L. lactis for the indicated time, and association of PrP C was determined by immunofluorescence microscopy. Hsp60 of B. abortus (D) or E. coli (E) is expressing on L. lactis surface. % PrP C positive refers to percentage of bacteria that showed costaining with PrP C . 100 bacteria were examined per coverslip. Data are the average of triplicate samples from three identical experiments, and the error bars represent the standard deviation.

Article Snippet: Anti-Hsp60 rabbit polyclonal antibody was obtained from MBL International Corporation.

Techniques: Incubation, Immunofluorescence, Microscopy, Bacteria, Staining, Labeling, In Vitro, Fluorescence, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation