sphingosine kinase 1 inhibitor ski5c  (Santa Cruz Biotechnology)

Journal: Lipids in health and disease

Article Title: A comprehensive multiomics approach reveals that high levels of sphingolipids in cardiac cachexia adipose tissue are associated with inflammatory and fibrotic changes.

doi: 10.1186/s12944-023-01967-0

Figure Lengend Snippet: Fig. 11 Metabolic processes of Cer and S1P in adipose tissue and mechanisms contributing to adipose tissue remodelling. SMase facilitates the conversion of SM into Cer. CDase is responsible for catalysing the conversion of Cer into Sph. S1P can be attributed to the enzymatic activity of SPHK. S1P can in turn be reversibly dephosphorylated by SPP/LPP to regenerate Sph, which is then converted back to Cer. Ceramide kinase (CerK) catalyzes Cer to produce CerP (C1P), which can in turn be reversibly dephosphorylated by CPP/LPP to regenerate Cer, which is then converted back to SM. Elevated levels of Cer and S1P in adipose tissue lead to the convergence of monocytes from blood to adipose tissue and their differentiation into macrophages. Activation of the Tgf-β/Smad signalling pathway by macrophages causes increased secretion of collagen and α-SMA by fibroblasts, resulting in excessive deposition of extracellular matrix. At the same time, macrophages aggregated to and infiltrated the adipose tissue, causing an inflammatory response in the adipose tissue. Adipose tissue inflammation-fibrosis interactions result in adipose tissue remodelling and dysfunction

Article Snippet: The membranes were then incubated in 5% skim milk at 25 °C for 1 h. Subsequently, the membranes were washed and incubated with the following primary antibodies overnight at 4 °C: β-actin (Cat No. 20536–1-AP, Proteintech, Wuhan, China), sphingosine kinase (SPHK) (1:1000, Cat No. 10670–1-AP, Proteintech, Wuhan, China), sphingosine 1-phosphate receptor 1 (S1PR1) (1:1000, Cat No. 55133–1-AP, Proteintech, Wuhan, China) and sphingosine 1-phosphate receptor 2 (S1PR2) (1:1000, Cat No. ab235919, Abcam, Cambridge, United Kingdom).

Techniques: Activity Assay, Activation Assay

Journal: Cell reports

Article Title: EGF-mediated suppression of cell extrusion during mucosal damage attenuates opportunistic fungal invasion

doi: 10.1016/j.celrep.2021.108896

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphingosine kinase 1 inhibitor (Ski5C) , Santa Cruz , SC-288340.

Techniques: Recombinant, Purification, Western Blot, Hybridization, Amplification, Gene Expression, Mutagenesis, Control, Software

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 by specific inhibitor DMS causes decrease in S1P level. ( A ) NBD-Sph and NBD-S1P are localized to host membrane and parasite. Infected erythrocytes was resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) and incubated with NBD-sphingosine (1 μM) for at least 15 min at 37 °C. The Infected erythrocytes were imaged by fluorescence using a 100 × oil objective on a Nikon Ti2 microscope. Schematic illustration of S1P and NBD-S1P synthesis and uptake in parasite. Erythrocytes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine kinase (SphK-1) to NBD-S1P, respectively. Parasites uptake NBD-S1P from erythrocytes. White arrow indicated the erythrocyte chosen for better depiction of NBD-S1P level as in form of zoomed-in images. ( B ) Bar graph depicts ELISA-based S1P quantification in infected and uninfected erythrocytes. ( C (i) ) Bar graph denotes the relative level of NBD-S1P. Uninfected erythrocytes, infected erythrocyte pre-treated with DMS were mixed with NBD-sphingosine (NBD-Sph) and incubated for the 1 h at 37 degree. For NBD-S1P quantification, the supernatant and cell pellets were transferred to 1.5-mL tubes containing methanol/chloroform (MeOH/CHCl3) mixture. NH4OH solution and CHCl3 were added to this mixture in a stepwise manner. After centrifugation, NBD-S1P and NBD-sphingosine were separated into aqueous (upper) and organic (lower) phases in the alkaline condition, respectively. The aqueous phases were transferred to a black 96-well plate and the fluorescence of the sample was measured. ( C (ii) ) Bar graph denotes the relative level of NBD-S1P in host free parasite post treated and host free parasites pre-treated with DMS, mixed with NBD-sphingosine (NBD-Sph) and incubated for the 1 h at 37 degree Celsius. Giemsa stained image depict the Uninfected erythrocytes and infected erythrocytes of asexual blood stage (ABS) and saponin lysed/Host free parasites. Three independent experiment have been performed, n = 3, ∗ p ≤ 0.05.

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Centrifugation, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 by specific inhibitor DMS causes decrease in SphK-1 protein level and phosphorylation. ( A ) Confocal micrographs demonstrate altered host SphK-1 level in mixed-stage parasite culture following treatment with DMS (14 µM). Bar graph denotes the differential mean fluorescence intensity (MFI) denoting host SphK-1 level in infected and uninfected erythrocytes following DMS treatment. The supportive immunofluorescence micrograph was incorporated in Supplementary Fig. . ( B ) The representative uncropped immunoblots show phosphorylation status of SphK-1 and its level in treated and untreated cells. Total cell lysates probed with GAPDH was used as a loading control. The graph represents fold change in the band intensity in individual lanes for two individual experiments. Three independent experiment have been performed, n = 3, ∗ p ≤ 0.05.

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Inhibition, Fluorescence, Infection, Immunofluorescence, Western Blot

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 blocks parasite growth and progression. ( A ) Parasite-infected erythrocytes mixed with erythrocytes to a final parasitemia of 1% and final hematocrit of 2% and incubated with S1P at different concentration (0–8 µM) for 48 h. ( B ) Percentage inhibition of P. falciparum growth was evaluated at different DMS concentrations (0-40 μM), as presented in the bar graph. Three independent experiments were performed in triplicates with 96-well plates using a SYBR-green assay. The IC50 value was determined as 14 µM for the parasite growth inhibition with DMS. ( C ) Visualization of stage specific inhibition of P. falciparum progression following DMS treatment was depicted by light microscopic images of Giemsa stained ring, trophozoite and schizont stages. ( D ) Scanning electron micrographs of DMS-treated infected and uninfected erythrocytes represented comparative morphometric analysis. Scale bars = 5 μm. Three biological replicates have been performed n = 3, ∗ p ≤ 0.05.

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Inhibition, Infection, Incubation, Concentration Assay, SYBR Green Assay, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 results in reduced glucose uptake and lactate production. ( A ) Evaluation of 2-NBDG uptake in parasite infected erythrocytes following DMS treatment by live-cell imaging. Respective MFI of individual infected erythrocytes were plotted against individual untreated cells. Three independent experiments have been done, n = 3, ∗∗ p ≤ 0.01. The representative live cell images were included in Supplementary Fig. ( B ) Representative histograms depict changes in number of FITC positive population in flow cytometry analysis correlating to 2-NBDG uptake following DMS treatment in infected erythrocytes. ( C ) Quantification of lactate in uninfected and infected erythrocytes treated with DMS (14 μM), was represented as changes in relative intensities. ( D ) Evaluation of lactate levels in asexual blood stages following DMS treatment, was plotted as percentage fold change. ( E ) Trophozoites and saponized/host-free trophozoites were used for experiment. Representative bar graph displayed changes in lactate levels as relative intensities for trophozoites and saponized/host-free parasites. The graph represents data for two individual experiments ∗ p ≤ 0.05.

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Inhibition, Infection, Live Cell Imaging, Flow Cytometry

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Translocation of GAPDH in parasite infected erythrocytes upon host SphK-1 inhibition. ( A ) Confocal micrographs demonstrate GAPDH level and localization in both DMS-treated and untreated infected erythrocytes. ( B ) Detection of GAPDH in both membrane and cytosolic fractions of DMS-treated infected erythrocytes. Total cell lysate was used as a loading control. Change in the level was plotted as relative intensities of bands detected in different cellular fractions. Three independent experiments have been done, n = 3, ∗ p ≤ 0.05. ( C ) Antimalarial effects of S1P, THI, DMS. Groups of BALB/c mice (n = 3 per group) were inoculated with 1 × 10 8 parasitized erythrocytes of pbANKA by IP injection. Post 48 h of infection; DMS was administered by intraperitoneal route at 4 µg/kg; oral administration of 2-Acetyl-4-tetrahydroxybutyl imidazole (THI) was done at 4 µg/kg and S1P was injected intravenously at a concentration of 0.4ug/kg. Untreated control mice received 10 ml/kg of DW. The treatment was carried out for 3 consecutive days (days 0–3). Every day parasitemia was determined by microscopic examination of Giemsa stained thin blood smears. Comparative analysis among the groups, namely; DMS, THI and S1P treated mice with untreated mice done with One-way ANOVA followed by Tukey multiple comparison test, which revealed significant differences between DMS treated and untreated mice with p values, p < 0.0001****, not significant (ns).

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Translocation Assay, Infection, Inhibition, Injection, Concentration Assay, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: In the proposed working model, life cycle of blood stage parasite is shown. In normal condition, S1P bind to deoxy-Hb and facilitates binding of deoxy-Hb to membrane and release of GAPDH; increased cytosolic GAPDH accelerates glycolysis and generate lactate which is a by-product of glycolysis and does not stall the growth of parasite. In case of DMS-mediated inhibition of host SphK-1, S1P level gets reduced, leading to altered binding with deoxy-Hb. Thus, it does not facilitate GAPDH to cytosol due to which glycolysis is suppressed leading to retarded parasite growth.

Article Snippet: Slides were probed with anti-SphK-1 (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:500) mouse antibodies in blocking buffer at RT for 1 h. After washing, slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Molecular Probes, USA, 1:500) and Alexa Fluor 488 conjugated goat anti-mouse IgG (Molecular Probes, USA, 1:500) at RT for 1 h. After washing, the slides were mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA), viewed on a Nikon A1-R confocal microscope and Olympus confocal microscopy.

Techniques: Binding Assay, Inhibition

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 by specific inhibitor DMS causes decrease in S1P level. ( A ) NBD-Sph and NBD-S1P are localized to host membrane and parasite. Infected erythrocytes was resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) and incubated with NBD-sphingosine (1 μM) for at least 15 min at 37 °C. The Infected erythrocytes were imaged by fluorescence using a 100 × oil objective on a Nikon Ti2 microscope. Schematic illustration of S1P and NBD-S1P synthesis and uptake in parasite. Erythrocytes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine kinase (SphK-1) to NBD-S1P, respectively. Parasites uptake NBD-S1P from erythrocytes. White arrow indicated the erythrocyte chosen for better depiction of NBD-S1P level as in form of zoomed-in images. ( B ) Bar graph depicts ELISA-based S1P quantification in infected and uninfected erythrocytes. ( C (i) ) Bar graph denotes the relative level of NBD-S1P. Uninfected erythrocytes, infected erythrocyte pre-treated with DMS were mixed with NBD-sphingosine (NBD-Sph) and incubated for the 1 h at 37 degree. For NBD-S1P quantification, the supernatant and cell pellets were transferred to 1.5-mL tubes containing methanol/chloroform (MeOH/CHCl3) mixture. NH4OH solution and CHCl3 were added to this mixture in a stepwise manner. After centrifugation, NBD-S1P and NBD-sphingosine were separated into aqueous (upper) and organic (lower) phases in the alkaline condition, respectively. The aqueous phases were transferred to a black 96-well plate and the fluorescence of the sample was measured. ( C (ii) ) Bar graph denotes the relative level of NBD-S1P in host free parasite post treated and host free parasites pre-treated with DMS, mixed with NBD-sphingosine (NBD-Sph) and incubated for the 1 h at 37 degree Celsius. Giemsa stained image depict the Uninfected erythrocytes and infected erythrocytes of asexual blood stage (ABS) and saponin lysed/Host free parasites. Three independent experiment have been performed, n = 3, ∗ p ≤ 0.05.

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Centrifugation, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 by specific inhibitor DMS causes decrease in SphK-1 protein level and phosphorylation. ( A ) Confocal micrographs demonstrate altered host SphK-1 level in mixed-stage parasite culture following treatment with DMS (14 µM). Bar graph denotes the differential mean fluorescence intensity (MFI) denoting host SphK-1 level in infected and uninfected erythrocytes following DMS treatment. The supportive immunofluorescence micrograph was incorporated in Supplementary Fig. . ( B ) The representative uncropped immunoblots show phosphorylation status of SphK-1 and its level in treated and untreated cells. Total cell lysates probed with GAPDH was used as a loading control. The graph represents fold change in the band intensity in individual lanes for two individual experiments. Three independent experiment have been performed, n = 3, ∗ p ≤ 0.05.

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Inhibition, Fluorescence, Infection, Immunofluorescence, Western Blot

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 blocks parasite growth and progression. ( A ) Parasite-infected erythrocytes mixed with erythrocytes to a final parasitemia of 1% and final hematocrit of 2% and incubated with S1P at different concentration (0–8 µM) for 48 h. ( B ) Percentage inhibition of P. falciparum growth was evaluated at different DMS concentrations (0-40 μM), as presented in the bar graph. Three independent experiments were performed in triplicates with 96-well plates using a SYBR-green assay. The IC50 value was determined as 14 µM for the parasite growth inhibition with DMS. ( C ) Visualization of stage specific inhibition of P. falciparum progression following DMS treatment was depicted by light microscopic images of Giemsa stained ring, trophozoite and schizont stages. ( D ) Scanning electron micrographs of DMS-treated infected and uninfected erythrocytes represented comparative morphometric analysis. Scale bars = 5 μm. Three biological replicates have been performed n = 3, ∗ p ≤ 0.05.

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Inhibition, Infection, Incubation, Concentration Assay, SYBR Green Assay, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Inhibition of host SphK-1 results in reduced glucose uptake and lactate production. ( A ) Evaluation of 2-NBDG uptake in parasite infected erythrocytes following DMS treatment by live-cell imaging. Respective MFI of individual infected erythrocytes were plotted against individual untreated cells. Three independent experiments have been done, n = 3, ∗∗ p ≤ 0.01. The representative live cell images were included in Supplementary Fig. ( B ) Representative histograms depict changes in number of FITC positive population in flow cytometry analysis correlating to 2-NBDG uptake following DMS treatment in infected erythrocytes. ( C ) Quantification of lactate in uninfected and infected erythrocytes treated with DMS (14 μM), was represented as changes in relative intensities. ( D ) Evaluation of lactate levels in asexual blood stages following DMS treatment, was plotted as percentage fold change. ( E ) Trophozoites and saponized/host-free trophozoites were used for experiment. Representative bar graph displayed changes in lactate levels as relative intensities for trophozoites and saponized/host-free parasites. The graph represents data for two individual experiments ∗ p ≤ 0.05.

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Inhibition, Infection, Live Cell Imaging, Flow Cytometry

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: Translocation of GAPDH in parasite infected erythrocytes upon host SphK-1 inhibition. ( A ) Confocal micrographs demonstrate GAPDH level and localization in both DMS-treated and untreated infected erythrocytes. ( B ) Detection of GAPDH in both membrane and cytosolic fractions of DMS-treated infected erythrocytes. Total cell lysate was used as a loading control. Change in the level was plotted as relative intensities of bands detected in different cellular fractions. Three independent experiments have been done, n = 3, ∗ p ≤ 0.05. ( C ) Antimalarial effects of S1P, THI, DMS. Groups of BALB/c mice (n = 3 per group) were inoculated with 1 × 10 8 parasitized erythrocytes of pbANKA by IP injection. Post 48 h of infection; DMS was administered by intraperitoneal route at 4 µg/kg; oral administration of 2-Acetyl-4-tetrahydroxybutyl imidazole (THI) was done at 4 µg/kg and S1P was injected intravenously at a concentration of 0.4ug/kg. Untreated control mice received 10 ml/kg of DW. The treatment was carried out for 3 consecutive days (days 0–3). Every day parasitemia was determined by microscopic examination of Giemsa stained thin blood smears. Comparative analysis among the groups, namely; DMS, THI and S1P treated mice with untreated mice done with One-way ANOVA followed by Tukey multiple comparison test, which revealed significant differences between DMS treated and untreated mice with p values, p < 0.0001****, not significant (ns).

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Translocation Assay, Infection, Inhibition, Injection, Concentration Assay, Staining

Journal: Scientific Reports

Article Title: Erythrocyte sphingosine kinase regulates intraerythrocytic development of Plasmodium falciparum

doi: 10.1038/s41598-020-80658-7

Figure Lengend Snippet: In the proposed working model, life cycle of blood stage parasite is shown. In normal condition, S1P bind to deoxy-Hb and facilitates binding of deoxy-Hb to membrane and release of GAPDH; increased cytosolic GAPDH accelerates glycolysis and generate lactate which is a by-product of glycolysis and does not stall the growth of parasite. In case of DMS-mediated inhibition of host SphK-1, S1P level gets reduced, leading to altered binding with deoxy-Hb. Thus, it does not facilitate GAPDH to cytosol due to which glycolysis is suppressed leading to retarded parasite growth.

Article Snippet: After washing, blots were incubated for 1 h with anti-SphK-1 (1:3000), anti-Phospho-SphK-1 (Ser225) (Invitrogen, Carlsbad, CA, USA, 1:1000) rabbit and anti-GAPDH (Invitrogen, Carlsbad, CA, USA, 1:10,000) mouse antibodies in blocking buffer.

Techniques: Binding Assay, Inhibition