rabbit anti gbf1  (Proteintech)

Journal: mSphere

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

doi: 10.1128/msphere.00770-23

Figure Lengend Snippet: Arf1 and GBF1 are uncoupled in the T. gondii PV. ( A ) Representative confocal immunofluorescence images of uninfected and Tg-mCh (red)-infected HeLa cells at 4, 24, and 48 hpi. Cells were stained with anti-GBF1 (green) and nuclei were stained with Hoechst (blue). ( B ) Quantification of T. gondii PVs with at least one punctum of GBF1 at 4 (gray), 24 (blue), and 48 (green) hpi. Data represent mean ± SEM, n = 3 biological replicates analyzing >50 PVs for each condition. ( C ) Assay schematic showing drug administration at 21 hpi and analysis at 24 hpi. Representative confocal immunofluorescence images of uninfected and Tg-mCh (red)-infected HeLa cells overexpressing Arf1-HA and treated with DMSO, 3.5 µM golgicide A (GCA), or 1.0 µM brefeldin A (BFA). Cells were stained with anti-HA (green), anti-GM130 (magenta), and nuclei were stained with Hoechst (blue). ( D ) Quantification of T. gondii PVs with at least one punctum of Arf1 at 24 hpi after treatment with DMSO (gray), GCA (blue), or BFA (green). Data represent the percentage of T. gondii PVs with at least one punctum of Arf1 compared to the DMSO control ±SEM, n = 3 biological replicates analyzing >50 PVs for each condition. P -values display one-way ANOVA with Dunnett’s multiple comparison test for each condition compared to DMSO. ns = non-significant. ( E ) Representative confocal immunofluorescence images of uninfected and Tg-mCh (red)-infected HeLa cells overexpressing Arf1-HA at 32 hpi. Cells were stained with anti-HA (green), anti-GBF1 (magenta), and nuclei were stained with Hoechst (blue). 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

Journal: Nature Communications

Article Title: TBC1D23 mediates Golgi-specific LKB1 signaling

doi: 10.1038/s41467-024-46166-2

Figure Lengend Snippet: a Volcano plot of quantitative analysis of phosphopeptides identified by MS. Blue or red: peptides with fold-change (TBC1D23 KO:WT) < 0.67 ( p < 0.01, t-test); green or purple: peptides with fold-change (TBC1D23 KO:WT) > 1.5 ( p < 0.01, t-test); red or purple: golgi-localized proteins; gray: no significant difference. b Combined analysis of phosphosites identified from AMPK DKO (AMPKα1/α2 double knockout) cells and TBC1D23 KO cells. The proportion of Golgi-localized proteins was determined. c Top 10 phosphosites with higher levels of phosphorylation in WT cells than TBC1D23 KO cells, ranked by p value. Golgi-localized proteins were shown in red color. d , e Constructs encoding Flag-tagged ARF1 WT ( d ) or S147A mutant ( e ) were transfected into WT or TBC1D23 KO HEK293T cells. 24 h later, cells glucose starved for 2 h were collected and subjected to immunoprecipitation using Flag beads and immunoblotting. The graph showed ratios of bound pST to Flag quantified by densitometry using Image J software. f WT and TBC1D23 KO HEK293T were glucose starved for the indicated time, and cell lysates were subjected to immunoblotting. The graph shows the levels of pGBF1 quantified by densitometry using Image J software and normalized to GBF1. g , h WT and TBC1D23 KO HepG2 cells were treated with or without 10 mM metformin for 4 h. The cells were stained with an anti-GM130 (Golgi marker) antibody. Representative confocal images ( g ) and quantitation of Golgi elements in cells as treated in ( g ) ( h , left panel). Scale bar, 10 μm. The ratio between the total area occupied by GM130 and DAPI was also used to indicate Golgi disassembly ( h , right pannel). Each dot in the figure represents the ratio GM130 area/nucleus area from one cell. Similar results were obtained in three independent experiments ( d – h ). Results are presented as mean ± SD ( d – h ). P values were determined by unpaired two-tailed t test ( a , c , d , e ), or by two-way ANOVA, followed by Sidak ’ s test ( f ), or by Scheirer-Ray-Hare Test ( h ). Source data are provided as a Source data file.

Article Snippet: Antibodies used in this study were as follows: rabbit polyclonal anti-TBC1D23 (Proteintech, 17002-1-AP, Western blot (WB) 1:1000), rabbit anti-LKB1(Cell Signaling Technology, 3047, WB 1:1000), rabbit anti-FAM21 (donated by Dr. Daniel D. Billadeauh, WB 1:1000), rabbit anti-AMPKα (Cell Signaling Technology, 2532, WB 1:1000), rabbit anti-phospho-AMPKα-Thr172 (Cell Signaling Technology, 2535, WB 1:1000), mouse anti-phospho-CaMKII-Thr286 (abcam, ab171095, WB 1:1000), rabbit anti-Flag (Proteintech, 20543-1-AP, WB 1:2000), mouse anti-Flag (Sigma-Aldrich, F1804, WB 1:2000, IF 1:300), rabbit anti-golgin-97 (Proteintech, 12640-1-AP, IF 1:200, WB 1:1000), mouse anti-LAMP1 (abcam, ab289548, WB 1:1000), mouse anti-TOM20 (santa cruz, sc-17764, WB 1:500), rat anti-HA (Roche, 11867423001,WB 1:2000), rabbit anti-GST (Proteintech, 10000-0-AP, WB 1:2000), mouse anti-GFP (Proteintech, 66002-1-Ig, WB 1:2000), rabbit anti-GBF1 (Proteintech, 25183-1-AP, WB 1:1000), rabbit anti-phospho-GBF1-Thr1337 (Immuno-Biological lab, 28065, WB 5 μg/mL), rabbit anti-GAPDH (Proteintech, 10494-1-AP, WB 1:2000), rabbit anti-beta actin (ABclonal, AC026, WB 1:2000), rabbit anti-mCherry (Proteintech, 26765-1-AP, WB 1:2000), rabbit anti-Phospho-(Ser/Thr) (Cell Signaling Technology, 9631, WB 1:1000), rabbit anti-GM130 (Abcam, 52649, IF 1:300), mouse anti-GM130 (BD, 610822, IF 1:300), rabbit anti-ZFPL1 (Invitrogen, PA5-53254, IF 1:300), mouse anti-TGN46 (Abcam, 50595, IF 1:300) and mouse anti-CIMPR (Bio-Rad, MCA2048, IF 1:300).

Techniques: Double Knockout, Construct, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Software, Staining, Marker, Quantitation Assay, Two Tailed Test