rabbit anti arf4  (Proteintech)

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Internalization of host Arfs into the T. gondii PV. Representative confocal immunofluorescence images of Tg-mCh (red)-infected HeLa cells overexpressing human Arf-HA fusion proteins. Cells were stained with an anti-HA antibody (green) and nuclei were stained with Hoechst (blue). ( A ) At 32 hpi, Arf1, Arf3, Arf4, Arf5, and Arf6 were observed within the T. gondii vacuole. Yellow arrows indicate the T. gondii PV. Insets (yellow boxes) of the T. gondii PV are shown to magnify Arf puncta. ( B ) Arf1, Arf3, Arf4, Arf5, and Arf6 were visualized in uninfected and Tg-mCh -infected HeLa cells at 4, 24, and 48 hpi. The enrichment of host Arfs at the PV was observed at 4 hpi (yellow arrows) which were internalized by the end of the lytic cycle at 48 hpi. ( C ) Quantification of T. gondii PVs with at least one punctum of host protein at 4 (gray), 24 (blue), and 48 (green) hpi. Data represent mean ± SEM; n = 3 biological replicates analyzing >50 PVs per each condition. ( D ) Plot showing the mean Arf fluorescent intensity values within the PV of infected cells at 48 hpi. n = 3 biological replicates analyzing a minimum of 20 PVs per replicate. P -values display one-way ANOVA with Dunnett’s multiple comparison test for each condition compared to Arf1. ** P < 0.01; **** P < 0.0001. Scale bars are 10 µm.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Immunofluorescence, Infection, Staining, Comparison

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Arf4 and GBF1 depletion impairs P. berghei liver stage development. ( A-C ) HuH7 cells were reverse transfected (15 nM) for 48 hours with a non-targeting scramble control (siCTRL, gray) or two pooled siRNAs targeting Arf4 (blue), GBF1 (green), and Arf1 (orange). ( A ) The relative mRNA levels of HuH7 cells treated with pooled siRNAs were determined by qRT-PCR. Samples were normalized to Hs 18S and compared to cells treated with siCTRL. ( B ) Forty-eight hours post-siRNA transfection, cells were infected with Pb- Luc sporozoites. HuH7 cell viability was assessed at 48 hpi using a CellTiter-Fluor assay. Data are normalized to siCTRL. ( C ) The parasite load was assessed at 48 hpi and normalized to cells treated with siCTRL. ( D-F ) HuH7 cells were reverse transfected (15 nM) for 48 hours with a non-targeting scramble control (siCTRL, gray) or two different siRNAs targeting Arf4 (1 and 2, blue) and GBF1 (1 and 2, green). ( D ) The relative mRNA levels of HuH7 cells treated with single siRNAs were determined by qRT-PCR. Samples were normalized to Hs 18S and compared to cells treated with siCTRL. ( E ) Forty-eight hours post-siRNA transfection, cells were infected with Pb- Luc sporozoites. HuH7 cell viability was assessed at 48 hpi using a CellTiter-Fluor assay. Data are normalized to siCTRL. ( F ) The parasite load was assessed at 48 hpi and normalized to cells treated with siCTRL. ( G-I ) Cells were reverse transfected with two pooled siRNAs (15 nM) for a non-targeting scramble control, Arf4, and GBF1. The ( G ) relative infection rate at 4 hpi, ( H ) relative infection rate at 48 hpi, and ( I ) PV size at 48 hpi were assessed for a non-targeting scramble control (siCTRL, gray), Arf4 (blue), and GBF1 (green). Data for the relative infection rates were normalized to cells treated with siCTRL. ( A-I ) Data represent mean ± SEM, n = 3 biological replicates. P -values display one-way ANOVA with Dunnett’s multiple comparison test for each condition compared to siCTRL. ns = non-significant * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Transfection, Quantitative RT-PCR, Infection, Comparison

Journal: mSphere

Article Title: Toxoplasma and Plasmodium associate with host Arfs during infection

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Arf4 and GBF1 are associated with the P. berghei PVM. ( A ) Representative confocal immunofluorescence microscopy images of P. berghei -infected HeLa cells overexpressing Arf1-HA at 24 and 48 hpi. Arf1-HA did not colocalize with the P. berghei PVM resident protein UIS4. Cells were stained with anti-HA (red), anti-USI4 (green), and nuclei were stained with Hoechst (blue). ( B and C ) P. berghei -infected HuH7 cells at 24 and 48 hpi were stained with ( B ) anti-Arf4 (red) or ( C ) anti-GBF1 (red). Cells were stained with anti-UIS4 (green) and nuclei were stained with Hoechst (blue). Scale bars are 10 µm. ( D and E ) The percentage of P. berghei vacuoles with Arf4 or GBF1 protein accumulation was assessed using Z-stacks of P. berghei -infected HuH7 cells at 48 hpi with Imaris software. ( D ) Protein accumulation to UIS4 was scored positive if the amount of Arf4 or GBF1 spots at the vacuole was statistically greater than a random distribution of protein within the host cell. ( E ) The attraction distance was calculated for vacuoles with a positive accumulation of Arf4 or GBF1 to the UIS4 surface. The value represents the distance from the UIS4 surface where there was a statistically greater accumulation of Arf4 or GBF1 than a random simulation. Data represent mean ± SEM, n = 3 biological replicates analyzing 20 PVs for each condition.

Article Snippet: Primary antibodies include mouse monoclonal anti-HA (Santa Cruz Cat# sc-7392, 1:400), rabbit monoclonal anti-HA (Cell Signaling Technology Cat# 3724, 1:400), rabbit monoclonal anti-GM130 (Abcam Cat# 52649, 1:200), rabbit monoclonal anti-GBF1 (Abcam Cat# 189512, 1:300), rabbit monoclonal anti-Arf4 (Abcam Cat# AB171746, 1:100), mouse monoclonal anti-V5 (Invitrogen Cat# R96025, 1:700), goat polyclonal anti-UIS4 (Antibodies.com Cat# A121573, 1:1000), and phalloidin-iFluor 647 (abclonal Cat# ab176759).

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Software