ptdins4p  (Echelon Biosciences)

Journal: Oncogene

Article Title: A novel cross-talk between CXCR4 and PI4KIIIα in prostate cancer cells.

doi: 10.1038/s41388-018-0448-0

Figure Lengend Snippet: PI4KIIIα regulates chemokine stimulated invasion of prostate cancer cells A) Representative immune fluorescence images showing punctate staining near invasive protrusions in PC3 cell lines displaying invasive phenotype. Merged images (merges) were obtained from overlays of the same image field from red, green and blue channels. PC3 RFP cells were stained with anti-PI4P antibody and Alexa Flour 488 (Green color) conjugated secondary antibody. PC3 CXCR4–2 cells have green fluorescence and were stained with anti-PI4P antibody and Alexa Flour 594 (Red color) conjugated secondary antibody. B) Quantification of the mean PI4P punctate fluorescence intensity (FI) performed on several point-like sources from 3 different fields shows a large increase in punctiform intensity in CXCR4–2 cells (p=0.008) vs control (RFP) PC3 cells. Shown are the means +/− S.E. (N=3) from 3 different fields; *, denotes statistical significance (p

Article Snippet: PC3 RFP and PC3 CXCR4–2 cells gown on coverslips were blocked and permeablized with 0.5% (w/v) saponin and incubated with mouse monoclonal anti-PI4P (Cat # Z-P004 from Echelon Biosciences); subsequently, Alexa Fluor 488 (for PC3 RFP cells) or 594 (for the PC3 CXCR4–2 or M-Luc2 cells) goat anti-mouse IgM(heavy chain) secondary antibody (Invitrogen) was used and were post-fixed with 2% formaldehyde and mounted on glass slide with antifade reagent containing DAPI.

Techniques: Fluorescence, Staining

Journal: Autophagy

Article Title: RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus

doi: 10.1080/15548627.2018.1532260

Figure Lengend Snippet: GcAVs contain PtdIns4P. (a) HeLa cells expressing EmGFP-OSBP-PH were infected with GAS for 4 h and immunostained with anti-TGOLN2 antibody. (b) HeLa cells expressing EmGFP-OSBP-PH and mCherry-LC3 were infected with GAS for 4 h. (c) HeLa cells expressing mCherry-LC3 were infected with GAS for 4 h and immunostained with anti-PtdIns4P antibody. Bars: 10 μm. NI, noninfected.

Article Snippet: The following primary antibodies were used: sheep anti-TGOLN2 (Novus Biological, NB110-40767), mouse anti-PtdIns4P (Echelon Biosciences, Z-P004), mouse anti-FLAG M2 (Sigma-Aldrich, A2220), mouse anti-GFP (GF200; Nacalai Tesque, 04363–24), rabbit anti-ACTB (13E5; Cell Signaling Technology, 4970), and rabbit anti-LC3B (D11; Cell Signaling Technology, 3868).

Techniques: Expressing, Infection

Journal: Autophagy

Article Title: RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus

doi: 10.1080/15548627.2018.1532260

Figure Lengend Snippet: PtdIns4P is important for autophagy during GAS infection. (a) Schematic representation of pathways for PtdIns3P and PtdIns4P synthesis and drugs targeting enzymes involved in these pathways. (b) HeLa cells expressing EmGFP-LC3 were treated with wortmannin (Wm) or PAO, and infected with GAS for 4 h. Bars: 10 μm. (c) HeLa cells expressing EmGFP-LC3 were treated with Wm or PAO at the indicated concentrations, and infected with GAS for 4 h. The percentages of GcAV-positive cells were quantified. (d) HeLa cells were treated with Wm (5 μM) or PAO (200 nM), and infected with GAS. Cells were analyzed by immunoblotting. (e) HeLa cells expressing EmGFP-LC3 were incubated with Wm (5 μM) and loaded with exogenous PtdIns4P, and infected with GAS for 4 h. The percentages of GcAV-positive cells were quantified. (f) HeLa cells treated with Wm or PAO at the indicated concentrations were infected with GAS for 2 and 6 h. The number of invading and surviving bacteria was measured in the GAS viability assay. Data in (c), (e), and (f) are the mean ± SD of 3 independent experiments. hpi, hours post infection.

Article Snippet: The following primary antibodies were used: sheep anti-TGOLN2 (Novus Biological, NB110-40767), mouse anti-PtdIns4P (Echelon Biosciences, Z-P004), mouse anti-FLAG M2 (Sigma-Aldrich, A2220), mouse anti-GFP (GF200; Nacalai Tesque, 04363–24), rabbit anti-ACTB (13E5; Cell Signaling Technology, 4970), and rabbit anti-LC3B (D11; Cell Signaling Technology, 3868).

Techniques: Infection, Expressing, Incubation, Viability Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

doi: 10.1073/pnas.1525719113

Figure Lengend Snippet: Plasma membrane PI4P production is critical for PS synthesis. ( A ) Lipidomic analysis of HEK293-AT1 cells treated with PI4K inhibitors for 30 h. Note the significant reduction in PS levels by the PI4KA inhibitor A1 (100 nM) but not by the PI4KB inhibitor

Article Snippet: Purified anti-PI4P monoclonal antibody (Z-P004) was purchased from Echelon Biosciences.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

doi: 10.1073/pnas.1525719113

Figure Lengend Snippet: Control of PI4P transfer between the PM and the ER by ORP5 and ORP8 proteins and by PS overproduction. ( A ) Schematics of BRET analysis of PM PI4P. Here a tandem P4M domain of Legionella pneumophila (P4M-2x) was fused to the Luciferase to monitor PM PI4P

Article Snippet: Purified anti-PI4P monoclonal antibody (Z-P004) was purchased from Echelon Biosciences.

Techniques: Bioluminescence Resonance Energy Transfer, Luciferase

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

doi: 10.1073/pnas.1525719113

Figure Lengend Snippet: PI4KA inhibition reduces PM PS levels, and PS overproduction affects PI4P levels. (A ) Schematics of the principles of quantification of PM-localized PS by BRET analysis. The PS-binding LacC2 domain fused to S-luciferase excites the PM-targeted Venus in

Article Snippet: Purified anti-PI4P monoclonal antibody (Z-P004) was purchased from Echelon Biosciences.

Techniques: Inhibition, Bioluminescence Resonance Energy Transfer, Binding Assay, Luciferase

Journal: Nature Communications

Article Title: Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking

doi: 10.1038/s41467-019-11056-5

Figure Lengend Snippet: Schematic summary and model. a The diagram schematically summarizing the cellular function of Dopey1–Mon2 complex: it promotes the centrifugal (solid line with arrow) and inhibits the centripetal (dotted line with arrow) membrane trafficking and organelle positioning. b A schematic model explaining how Dopey1–Mon2 complex engages the membrane and kinesin-1. Mon2 is a constitutive homodimer and its dimerization is mediated by MEC at its C-terminus. Its N-terminal region interacts with PA (directly or indirectly). In contrast, Dopey1 is a monomer. However, two copies of Dopey1 can simultaneously bind to a Mon2 homodimer, resulting in the formation of a heterotetrameric complex. The interaction between Dopey1 and Mon2 is through Mon2’s MEC and Dopey1’s central region. DEN at the N-terminus of Dopey1 interacts with KLC2’s TPR domain via the WD-motif, while DEC at the C-terminus functions as a PI4P-binding module. In Dopey1–Mon2 complex, Mon2 seems to dimerize Dopey1 for its high affinity binding to membrane-PI4P and KLC2 homodimer

Article Snippet: Mouse mAb against PI4P (#Z-P004)(1:100 for IF) was from Echelon Biosciences.

Techniques: Cell Function Assay, Binding Assay

Journal: Nature Communications

Article Title: Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking

doi: 10.1038/s41467-019-11056-5

Figure Lengend Snippet: Dopey1 is sufficient to interact with and recruit kinesin-1 to the membrane. a Dopey1 specifically interacts with Kif5b. b Dopey1(1–148) is sufficient to interact with KLC2. c W 34 is essential for DEN’s interaction with KLC2. d Dopey1–KLC2 interaction is enhanced by Mon2. Ratios of Dopey1 band intensity of the IP panel to that of the corresponding cell lysate panel were normalized and labeled in the top blot. IPs were similarly performed as in Fig. 1 . e The membrane recruitment of KLC2 requires PI4P and Dopey1 and is enhanced by Mon2. Liposomes with PI or PI4P were incubated with cell lysates expressing indicated protein(s) and pull-downs were immunoblotted. Values are normalized ratios of band intensities in pull-down panels to corresponding ones in cell lysate panels. Molecular weights (in kDa) are labeled. f , g The Golgi association of Kif5b decreases upon the knockdown of Dopey1 or Mon2. The experiment and quantification are similar to Fig. 2d, e . h – j Artificially tethering Dopey1 is sufficient to move peroxisomes to the cell periphery. A schematic diagram of fusion proteins is shown in ( h ). i Cells co-expressing indicated proteins were treated with 50 nM rapamycin followed by live cell imaging. The normalized gyradius is quantified in ( j ). k – p Particle analysis of peroxisomes with artificially tethered Dopey1. k Instantaneous velocity of a typical peroxisome tethered with indicated fusion protein. Segment speed, time, frequency, distance, and their corresponding centrifugal biases are shown in ( l – o ). p Statistical analysis of single peroxisome mean velocity. In each case, tracks were identified from time lapses of seven cells in three experiments. n indicates the number of cells ( g ), segments ( l , m , o ), or tracks ( n , p ). Scale bar, 10 µm; error bar, mean ± s.e.m.; P values are from t test (unpaired and two-tailed); N.S. not significant, * P ≤ 0.05, *** P ≤ 0.0005, **** P ≤ 0.00005, ***** P ≤ 0.000005. Dotted white lines indicate cell contours ( f , i ). HEK293T cells were used in a - e while HeLa cells were used in ( f , g , i– p ). Source data are provided as a Source Data file

Article Snippet: Mouse mAb against PI4P (#Z-P004)(1:100 for IF) was from Echelon Biosciences.

Techniques: Labeling, Incubation, Expressing, Live Cell Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking

doi: 10.1038/s41467-019-11056-5

Figure Lengend Snippet: Dopey1–Mon2 complex promotes the Golgi-to-PM but inhibits the reverse trafficking. a – c The Golgi exit of cargos and Golgi-to-PM trafficking require Dopey1 and Mon2. siRNA-treated cells expressing indicated RUSH reporter were subjected to the Golgi-to-PM trafficking assay (see Methods). Reporters arrived at the PM were surface-labeled by anti-GFP ( a ). Dotted white lines indicate cell contours. Golgi fractions were plotted in ( b , c ). d Mon2, Dopey1, Rab6, Kif5b, and PI4P can be found on membrane carriers involved in the Golgi-to-PM trafficking. Cells co-expressing indicated proteins were subjected to biotin and 20 °C treatment similar to a , and subsequently imaged live during 37 °C chase under a spinning disk confocal microscope. The region of interest is enlarged at the upper right. Arrows indicate colocalization. e – j Particle analysis of single carriers in the Golgi-to-PM trafficking. siRNA-treated cells expressing SBP-GFP-E-cadherin were subjected to treatment described in ( d ) except that imaging was conducted under the wide-field microscopy. See Supplementary Movie 3 for the control. e Instantaneous velocity of a typical carrier. Segment speed, time, frequency, distance, and their corresponding centrifugal biases are displayed in ( f – i ). j The statistical analysis of single carrier mean velocity. n, the number of segments in ( f , g , i or tracks in h , j ). In each case, tracks were identified from time lapses of eight cells in three experiments. k , l Depletion of Dopey1 or Mon2 accelerates the PM-to-Golgi trafficking. siRNA-treated cells expressing indicated CD8a-TGN-tails were subjected to the PM-to-Golgi trafficking assay (see Methods) and Golgi fractions were plotted against the chase time. Scale bar, 10 µm; error bar, mean ± s.e.m. In b , c , k and l , plots are representative results of three independent experiments; n indicates the number of cells analyzed. P values are from the t test (unpaired and two-tailed) comparison between indicated pairs ( f– j ) or between the knockdown and corresponding control ( b , c , k , l ). N.S. not significant, * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0005, **** P ≤ 0.00005, ***** P ≤ 0.000005. Source data are provided as a Source Data file

Article Snippet: Mouse mAb against PI4P (#Z-P004)(1:100 for IF) was from Echelon Biosciences.

Techniques: Expressing, Labeling, Microscopy, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking

doi: 10.1038/s41467-019-11056-5

Figure Lengend Snippet: Dopey1 specifically and directly interacts with PI4P. a – f The Golgi localization of Dopey1 and DEC, but not Mon2, is sensitive to PAO treatment. GFP-tdDEC transfected ( a , b ) or untransfected cells ( c – f ) were treated with 10 µM PAO followed by staining of indicated proteins. Dotted white lines demonstrate cell contours ( c , e ). In b , d , and f , the normalized Golgi intensity per cell was plotted against the time below the corresponding panel; error bar indicates mean ± s.e.m; n is the number of cells analyzed; results are representatives of three independent experiments. g , h The Golgi localization of Dopey1, but not Mon2, decreases upon the depletion of PI4KIIIβ. Indicated shRNAs were expressed using lentivirus and cells were stained for indicated proteins. The normalized Golgi intensity per cell is plotted at the right. Red dot, individual data point; error bar, mean ± s.d. from n = 3 independent experiments. i – m Dopey1 and DEC specifically interact with PI4P. Liposomes with 10% (m/m) indicated phosphoinositides were incubated with either the cell lysate expressing indicated fusion protein(s) ( i – l ) or purified recombinant protein ( m ). Proteins pulled down were analyzed by immunoblotting using indicated antibody. n , o Dimerized Dopey1 does not require Mon2 for its Golgi localization. Cells were subjected to control or Mon2 knockdown followed by transient expression of indicated construct. Endogenous Golgin97 (a Golgi marker) ( n ) or Golgin245 ( o ) was stained. Scale bar, 10 µm. Molecular weights (in kDa) are labeled in all gel blots. HeLa cells were used in ( a – h , n , o ), while HEK293T cells were used in ( i – l ). P values are from t test (unpaired and two-tailed). N.S. not significant, *** P ≤ 0.0005. Source data are provided as a Source Data file

Article Snippet: Mouse mAb against PI4P (#Z-P004)(1:100 for IF) was from Echelon Biosciences.

Techniques: Transfection, Staining, Incubation, Expressing, Purification, Recombinant, Construct, Marker, Labeling, Two Tailed Test