rabbit α gnpat  (Proteintech)

Journal: Science Advances

Article Title: Bacterial pathogens hijack host cell peroxisomes for replication vacuole expansion and integrity

doi: 10.1126/sciadv.adr8005

Figure Lengend Snippet: ( A ) Peroxisomes at the LCV are larger than those in the rest of the cell. Primary murine macrophages were challenged with WT bacteria, fixed, and visualized by fluorescence microscopy (as in ). Images were deconvoluted in three dimensions and the size of PMP70 puncta, based on the volume occupied in three-dimensional space were quantified (see Materials and Methods). ( B ) The relative abundance of peroxisomes at the LCV is similar to the rest of the cell. The number of PMP70 puncta from experiments in (A) was quantified, normalizing to the volume of the respective compartments. [(A) and (B)] Data are the combined measurements of two biological replicates, scoring 75 cells each. An asterisk indicates a two-way analysis of variance (ANOVA) with a post hoc two-tailed, nonparametric t test with Welch correction P < 0.0001. ( C ) L. pneumophila infection alters the abundance of peroxisomal proteins. THP-1 cells were infected for the indicated times and the amount of ACOX1, GNPAT, and AGPS in infected (I) cells was compared to uninfected (UI) cells by Western analysis (upper panel), and quantified (lower panel), normalizing to total protein (fig. S11B) and reported as fold change relative to 1 hour in uninfected cells. ( D ) Disruption of peroxisome lipid biosynthetic genes impairs L. pneumophila intracellular growth. THP-1 cells depleted of GNPAT or AGPS, based on Western analysis of whole-cell lysates (left panel), were infected with WT L. pneumophila and bacterial replication was quantified on the basis of recovered cfus from host cell lysates at 24 hours and normalized to the WT bacteria in WT host cells by the number of bacteria at 1 hpi. [(C) and (D)] Data are the mean ± SD of three biological replicates consisting of three technical replicates each. An asterisk indicates a Student’s t test P < 0.05 compared to uninfected cells at the same time point (C) or WT bacteria in WT host cells (D).

Article Snippet: Equal amounts of protein were examined by Western analysis, probing with the target specific antibody, rabbit α-PEX5 (1:500) (Proteintech), rabbit α-GNPAT (1:1000) (Proteintech), or mouse α-AGPS (1:500) (Santa Cruz) overnight at 4°C, followed by HRP-conjugated α-rabbit IgG (1:5000) (Sigma-Aldrich) or HRP-conjugated α-mouse IgG (1:5000) (Sigma-Aldrich), as appropriate.

Techniques: Bacteria, Fluorescence, Microscopy, Two Tailed Test, Infection, Western Blot, Disruption

Journal: Science Advances

Article Title: Bacterial pathogens hijack host cell peroxisomes for replication vacuole expansion and integrity

doi: 10.1126/sciadv.adr8005

Figure Lengend Snippet: ( A ) LCVs in macrophages lacking functional peroxisomes exhibit compact architecture. Wild-type (WT) immortalized murine macrophages (RAW264.7 cells) lacking either PEX19 or PEX5 or cells lacking PEX5 that were exogenously supplemented with palmitate (PA) were infected with L. pneumophila for 12 hours, fixed, and visualized by fluorescence microscopy. ( B ) LCVs in (A) were scored as spread (the majority of bacteria aligned end to end or at various angles) or compact (the majority of bacteria bundled together with their sidewalls juxtaposed to one another, with no bacteria extending beyond the boundary of a spherical shape) based on the organization of the bacteria in three-dimensional space . ( C ) Peroxisome defects lead to increased numbers of ruptured LCVs. Infected cells as in (A) were stained for L. pneumophila ( Lp ) and Galectin-3 (Gal3). ( D ) LCVs in (C) were scored for colocalization with Gal3. ( E ) Disruption of peroxisome lipid metabolic pathways limits LCV expansion and causes LCV destabilization. WT human monocyte–derived macrophages (THP-1 cells) lacking PEX5, GNPAT, or AGPS (Fig. 5B) were infected with L. pneumophila for 12 hours, fixed, and visualized by fluorescence microscopy. ( F ) LCVs in (E) were scored as spread or compact, as described in (B). ( G ) Gal3 colocalization with LCVs in (E) was quantified. [(B), (C), (F), (G)] Data are the mean ± SD of three biological replicates consisting of three technical replications each, scoring 50 to 100 vacuoles per technical replicate. An asterisk indicates a Student’s t test P < 0.05 compared to WT, unless otherwise indicated. ns, not significant.

Article Snippet: Equal amounts of protein were examined by Western analysis, probing with the target specific antibody, rabbit α-PEX5 (1:500) (Proteintech), rabbit α-GNPAT (1:1000) (Proteintech), or mouse α-AGPS (1:500) (Santa Cruz) overnight at 4°C, followed by HRP-conjugated α-rabbit IgG (1:5000) (Sigma-Aldrich) or HRP-conjugated α-mouse IgG (1:5000) (Sigma-Aldrich), as appropriate.

Techniques: Functional Assay, Infection, Fluorescence, Microscopy, Bacteria, Staining, Disruption, Derivative Assay